Depression gene

ABSTRACT

The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human depression predisposing gene, specifically the apoptotic protease activating factor 1 (APAF1) gene, some mutant alleles of which cause susceptibility to depression. More specifically, the invention relates to germline mutations in the APAF1 gene and their use in the diagnosis of predisposition to depression. The invention also relates to the prophylaxis and/or therapy of depression associated with a mutation in the APAF1 gene. The invention further relates to the screening of drugs for depression therapy. Finally, the invention relates to the screening of the APAF1 gene for mutations/alterations, which are useful for diagnosing the predisposition to depression.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/441,887 filed on May 26, 2006, which claims priority to U.S. patentapplication Ser. No. 10/646,396 filed on Aug. 22, 2003, now U.S. Pat.No. 7,052,853, which claims priority to U.S. Provisional ApplicationSer. Nos. 60/405,334, filed on Aug. 21, 2002; 60/428,513, filed Nov. 21,2002; and 60/442,492, filed Jan. 24, 2003, the contents of which areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledIHCINC_(—)003C1C1_SEQ.txt, created May 15, 2007, which is 18.5 Kb insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to depression. In particular, theinvention relates to a gene associated with depression and altered formsof the gene. The invention provides methods for predicting depression,predicting susceptibility to depression, and screening for drugs capableof treating depression.

2. Description of the Related Art

Depression is thought to affect around twenty million Americans everyyear. The economic impact of depression is difficult to estimate, butreports indicate that approximately 30 billion dollars was lost directlyand indirectly in 1990 as a result of the disease. Depression manifestsitself in many different ways including persistent sad mood, loss ofinterest or pleasure in once enjoyable activities, significant change inappetite or body weight, sleep disorders, physical slowing or agitation,loss of energy, feelings of worthlessness, inappropriate guilt,difficulty thinking, difficulty concentrating, malaise, and recurrentthoughts of death or suicide. The families and friends of depressedindividuals are often profoundly affected by the disease.

The present invention relates generally to depression. Morespecifically, the present invention relates to methods and materialsused to isolate and detect a human depression predisposing gene,specifically the apoptotic protease activating factor 1 (APAF1) gene,some mutant alleles of which cause susceptibility to depression. Morespecifically, the invention relates to germline mutations in the APAF1gene and their use in the diagnosis of predisposition to depression. Theinvention also relates to the prophylaxis and/or therapy of depressionassociated with a mutation in the APAF1 gene. The invention furtherrelates to the screening of drugs for depression therapy. Finally, theinvention relates to the screening of the APAF1 gene formutations/alterations, which are useful for diagnosing thepredisposition to depression.

Depression is typically diagnosed as major depressive disorder (unipolarmajor depression, bipolar disorder (manic-depressive illness), anddysthymic disorder (dysthymia). There are a number of subtypes of thesemajor categories of depression. Diagnosis of these mental disorders isbased on the Diagnostic and Statistical Manual of Mental Disorders,fourth edition (DSM-IV). American Psychiatric Association; Diagnosticand Statistical Manual of Mental Disorders, fourth edition (DSM-IV),Washington, D.C., American Psychiatric Press, 1994.

Major depression is associated with low mood, low energy and motivation,insomnia, and feelings of worthlessness and hopelessness. Bipolardisorder is a severe psychiatric disorder that affects approximately 1%of the world's population (Goodwin, F. K. and Jameson, K. R. (1990)Manic-Depressive Illness, Oxford Univ. Press, New York). It ischaracterized by extreme swings in mood between mania and depression.Mania is accompanied by euphoria, grandiosity, increased energy,decreased need for sleep, rapid speech, and risk taking. Psychosis canoccur in either state, and there is a 17% lifetime risk for suicide.Dysthymic disorder is considered a milder form of depression withsymptom similar to that of major depression.

The etiology of depression is currently unknown, but epidemiologicalstudies argue for a strong genetic component. Family studies indicate anapproximately 7-fold increase in risk to first-degree family members(Tsuang, M. T. and Faraone, S. V. (1990) The Genetics of Mood Disorders,Johns Hopkins Univ. Press, Baltimore). Twin studies find an average4-fold increase in risk to monozygotic vs. dizygotic twins. The mode ofgenetic transmission is unclear. Although some studies have supportedthe presence of autosomal dominant major loci (Spence, M. A. et al.(1995) Am. J. Med. Genet. 60:370 376; Rice, J. et al (1987) Arch. Gen.Psychiatry 44:441 447), it has also been argued that bipolar disorder isoligogenic with multiple loci of modest effect.

Although initial attempts at linkage studies met with inconsistentreplication (Egeland, J. A. et al. (1987) Nature 325:783 787; Kelsoe, J.R. et al. (1989) Nature 342:238 243; Baron, M. et al. (1987) Nature326:289 292; Baron, M. (1991) Soc. Biol. 38:179 188), more recently, theaccumulation of multiple studies of larger family sets has led to thereproducible identification of several genetic loci associated withdepression. These include 4p, 12q, 13q, 18, 21q, and Xq among others(Blackwood, D. H. et al. (1996) Nat. Genet. 12:427 430; Dawson, E. etal. (1995) Am. J. Med. Genet. 60:94 102; Detera-Wadleigh, S. D. et al.(1999) Proc. Natl. Acad. Sci. USA 96:5604 5609; Berrettini, W. H. et al.(1994) Proc. Natl. Acad. Sci. USA 91:5918 5921; Freimer, N. B. et al.(1996) Nat. Genet. 12:436 441; Straub, R. E. et al. (1994) Nat. Genet.8:291 296; Pekkarinen, P. et al. (1995) Genome Res. 5:105 115; Craddock,N. & Jones, I. (1999) J. Med. Genet. 36:585 594; Craddock, N. & Jones,I. (2001) Br. J. Psychiatry 41:s128-s133). Linkage between bipolardisorder and chromosome 12q23 12q24 has been reported (Green, E. K. etal. (2000) Am. J. Med. Genet. 96:545; Morissette, J. et al. (1999) Am.J. Med. Genet. 88: 567 587; Ewald, H. et al. (1998) Psychiatr. Genet.8:131 140 (1998); Degan, B. et al. (2001) Mol. Psychiatry 6:450 455;Detera-Wadleigh, S. D. et al. (1999) Am. J. Med. Genet. 88:255 259;Jacobsen, N. et al. (1996) Psych. Genet. 6:195 199; Rice, J. P. et al.(1997) Am. J. Med. Genet. 74:247 253).

In view of the importance of early diagnosis of depression, there is aneed to identify genes associated with depression for diagnostic andtherapeutic purposes.

SUMMARY OF THE INVENTION

This invention is based on the discovery of the first evidenceimplicating specific mutations in the APAF1 gene with susceptibility todepression. The inventors have discovered that mutations in APAF1 whichsegregate with major depression correlate to an enhancement in caspaseactivation in apoptosome reconstitution assays.

In a first aspect of the invention, a method for detecting in anindividual a susceptibility to depression is provided. Thus, the presentinvention provides methods for determining whether a subject is at riskfor developing depression due to a mutation in the APAF1 gene. Thismethod relies on the fact that mutations in the APAF1 have beencorrelated by the inventors with the disease. It will be understood bythose of skill in the art, given the disclosure of the invention thatsuch mutations are associated with a susceptibility to depression, thata variety of methods can be utilized to detect mutations in the APAF1gene, including the mutations disclosed herein, which are associatedwith a susceptibility to depression.

The method can include detecting, in a tissue of the subject, thepresence or absence of a polymorphism of the APAF1 gene. The detectionof a polymorphism in the APAF1 gene can include ascertaining theexistence of at least one of: a deletion of one or more nucleotides; anaddition of one or more nucleotides, a substitution of one or morenucleotides; a gross chromosomal rearrangement; an alteration in thelevel of a messenger RNA transcript; the presence of a non-wild typesplicing pattern of a messenger RNA transcript; a non-wild type level ofan APAF1 protein; and/or an aberrant level of an APAF1 protein.

For example, detecting the polymorphism can include (i) providing aprobe/primer comprised of an oligonucleotide which hybridizes to a senseor antisense sequence of an APAF1 gene or naturally occurring mutantsthereof, or 5′ or 3′ flanking sequences naturally associated with anAPAF1 gene; (ii) contacting the probe/primer to an appropriate nucleicacid containing sample; and (iii) detecting, by hybridization of theprobe/primer to the nucleic acid, the presence or absence of thepolymorphism; e.g. wherein detecting the polymorphism comprisesutilizing the probe/primer to determine the nucleotide sequence of anAPAF1 gene and, optionally, of the flanking nucleic acid sequences. Forinstance, the primer can be employed in a polymerase chain reaction(PCR), in a ligase chain reaction (LCR) or other amplification reactionsknown to a skilled artisan. In alternate embodiments, the level of anAPAF1 protein is detected in an immunoassay using an antibody which isspecifically immunoreactive with the APAF1 protein. In anotherembodiment, antibodies specific to APAF1 mutants are used to determinethe APAF1 for diagnostic purposes.

In a second aspect of the invention, compounds that are agonists orantagonists of a normal (functional) APAF1 bioactivity and their use inpreventing or treating depression are provided. For example, toameliorate disease symptoms involving insufficient expression of anAPAF1 gene and/or inadequate amount of functional APAF1 bioactivity in asubject, a gene therapeutic (comprising a gene encoding a functionalAPAF1 protein) or a protein therapeutic (comprising a functional APAF1protein or fragment thereof) can be administered to the subject.Alternatively, agonists or antagonists of APAF1 function (wild-type ormutant) or an APAF1 receptor or a receptor for fragments of APAF1 can beadministered.

In a third aspect of the invention, compounds that are antagonists of adisease causing APAF1 bioactivity and their use in preventing ortreating depression are provided. For example, to ameliorate diseasesymptoms involving expression of a mutant APAF1 gene or aberrantexpression of a normal APAF1 gene in a subject, a therapeuticallyeffective amount of an antisense, ribozyme, siRNA, or triple helixmolecule to reduce or prevent gene expression may be administered to thesubject. Alternatively, to ameliorate disease symptoms involving theregulation via an APAF1 protein or APAF1 protein fragments of anupstream or downstream element in an APAF1 mediated biochemical pathway(e.g., signal transduction), a therapeutically effective amount of anagonist or antagonist compound (e.g., small molecule, peptide,peptidomimetic, protein or antibody) that can prevent normal binding ofthe wild-type APAF1 protein, can induce a therapeutic effect.

In fourth aspect of the invention, assays, e.g., for screening testcompounds to identify antagonists (e.g. inhibitors), or alternatively,agonists (e.g. potentiators), of an interaction between an APAF1 proteinand, for example, a protein or nucleic acid that binds to the APAF1protein or fragments of APAF1 are provided. An exemplary method includesthe steps of (i) combining an APAF1 polypeptide or bioactive fragmentsthereof, an APAF1 target molecule (such as an APAF1 ligand or nucleicacid), and a test compound, e.g., under conditions wherein, but for thetest compound, the APAF1 protein and APAF1 target molecule are able tointeract; and (ii) detecting the formation of a complex which includesthe APAF1 protein and the target molecule either by directlyquantitating the complex or by measuring inductive effects of the APAF1protein or fragments of APAF1 protein. A statistically significantchange, such as a decrease, in the interaction of the APAF1 and APAF1target molecule in the presence of a test compound (relative to what isdetected in the absence of the test compound) is indicative of amodulation (e.g., inhibition or potentiation of the interaction betweenthe APAF1 protein or fragments of the APAF1 protein and the targetmolecule).

In a fifth aspect, the present invention provides methods for modulatingthe transcription of certain genes in a cell by modulating APAF1bioactivity, (e.g., by potentiating or disrupting an APAF1 bioactivity).In general, whether carried out in vivo, in vitro, or in situ, themethod comprises treating the cell with an effective amount of an APAF1therapeutic (agonist or antagonist of an APAF1 bioactivity) so as toalter, relative to the cell in the absence of treatment, the level oftranscription of certain genes. Accordingly, the method can be carriedout with APAF1 therapeutics such as peptide and peptidomimetics or othermolecules identified in the above-referenced drug screens which agonizeor antagonize the effects of an APAF1 bioactivity (e.g. transcription)of a gene which is regulated by an APAF1 protein. Other APAF1therapeutics include antisense or siRNA constructs for inhibitingexpression of APAF1 proteins, and dominant negative mutants of APAF1proteins which competitively inhibit interactions between ligands (e.g.proteins) and nucleic acids upstream and downstream of the wild-typeAPAF1 protein.

In a sixth aspect, the invention relates to isolated nucleic acidsencoding an altered APAF1. In particular, the invention provides anisolated altered APAF1 nucleic acid, having one or more of the followingalterations (in reference to nucleotides 1 to 3744 set forth SEQ ID: 1):(a) the C at nucleotide position 1350 is substituted with G, or acomplement thereof; (b) the A at nucleotide position 1394 is substitutedwith G, or a complement thereof; (c) the G at nucleotide position 2329is substituted with A, or a complement thereof; (d) the A at nucleotideposition 2345 is substituted with C, or a complement thereof; (e) the Aat nucleotide position 2857 is substituted with G, or a complementthereof; (f) a T is inserted after nucleotide position 1299, or acomplement thereof; (g) the T at nucleotide position 1244 is substitutedwith C, or a complement thereof; (h) the C at nucleotide position 1070is substituted with T, or a complement thereof; (i) the C at nucleotideposition 1437 is substituted with G, or a complement thereof; and (j)the A at nucleotide position 1874 is substituted with C, or a complementthereof.

In a seventh aspect, the invention provides a nucleic acid probespecifically hybridizable to a human altered APAF1 and not to thecorresponding wild-type DNA. According to one embodiment of this aspectof the invention, the altered APAF1 comprises an alteration of SEQ IDNO:1 selected from C1350G, A1394G, G2329A, A2345C, A2857G, insertion ofa T after nucleotide position 1299, T1244C, C1070T, C1437G, A1874C orcomplements thereof.

In an eighth aspect, the invention provides a method for diagnosing analteration which causes depression by hybridizing a probe to an altered(mutant) APAF1 nucleic acid in a patient's sample of DNA or RNA understringent conditions which allows hybridization of the probe to nucleicacid comprising the alteration but prevents hybridization of the probeto a wild-type nucleic acid. The presence of a hybridization signalindicates the presence of the alteration. In a preferred embodiment ofthis aspect of the invention, the method is performed using nucleic acidmicrochip technology.

In a ninth aspect, the invention provides an isolated altered APAF1polypeptide. According to this aspect of the invention, the isolatedpolypeptide has (in reference to SEQ ID NO:2): (a) the Cys at position450 substituted with Trp; (b) the Gln at position 465 substituted withArg; (c) the Glu at position 777 substituted with Lys; (d) the Asn atposition 782 substituted with Thr; (e) the Thr at position 953substituted with Ala; (f) the Leu at position 415 substituted with Pro;(g) the Ser at position 357 substituted with Leu; (h) the Asp atposition 479 substituted with Glu; or (i) the Glu at position 625substituted with Ala. In one embodiment of this aspect of the invention,the invention provides a protein molecule comprising the amino acids setforth in SEQ ID NO:3. In another embodiment of this aspect of theinvention an antibody capable of binding the altered polypeptide butincapable of binding a wild-type APAF1 polypeptide is provided.

In a tenth aspect, the invention provides a method for detecting analteration in APAF1 that is associated with depression in a human.Accordingly, the method comprises analyzing an APAF1 gene or an APAF1gene expression product from cells or tissue of a human. In someembodiments, the mutation is detected by immunoblotting,immunocytochemistry, assaying for binding interactions between the geneproduct isolated from the tissue and a binding partner capable ofspecifically binding the polypeptide expression product of a mutantallele and/or a binding partner for the polypeptide, or assaying for theinhibition of biochemical activity of the binding partner. In anotherembodiment of this aspect of the invention, the method involvescomparing the sequence of a subject APAF1 gene with the sequence of oneor more wild-type APAF1 gene sequences. According to other embodimentsof this aspect of the invention, the mutation can be detected by anymethod including: (a) hybridizing a probe specific for one of thealterations to RNA isolated from the human sample and detecting thepresence of a hybridization product, wherein the presence of the productindicates the presence of the alteration in the sample; (b) hybridizinga probe specific for one of the alterations to cDNA made from RNAisolated from the sample and detecting the presence of a hybridizationproduct, wherein the presence of the product indicates the presence ofthe alteration in the sample; (c) hybridizing a probe specific for oneof the alterations to genomic DNA isolated from the sample and detectingthe presence of a hybridization product, wherein the presence of theproduct indicates the presence of the alteration in the sample; (d)amplifying all or part of the gene in the sample using a set of primersto produce amplified nucleic acids and sequencing the amplified nucleicacids; (e) amplifying part of the gene in the sample using a primerspecific for one of the alterations and detecting the presence of anamplified product, wherein the presence of the product indicates thepresence of the alteration in the sample; (f) molecularly cloning all orpart of the gene in the sample to produce a cloned nucleic acid andsequencing the cloned nucleic acid; (g) amplifying the gene to produceamplified nucleic acids, hybridizing the amplified nucleic acids to aDNA probe specific for one of the alterations and detecting the presenceof a hybridization product, wherein the presence of the productindicates the presence of the alteration; (h) forming single-strandedDNA from a gene fragment of the gene from the human sample andsingle-stranded DNA from a corresponding fragment of a wild-type gene,electrophoresing the single-stranded DNAs on a non-denaturingpolyacrylamide gel and comparing the mobility of the single-strandedDNAs on the gel to determine if the single-stranded DNA from the sampleis shifted relative to wild-type and sequencing the single-stranded DNAhaving a shift in mobility; (i) forming a heteroduplex consisting of afirst strand of nucleic acid selected from the group consisting of agenomic DNA fragment isolated from the sample, an RNA fragment isolatedfrom the sample and a cDNA fragment made from mRNA from the sample and asecond strand of a nucleic acid consisting of a corresponding humanwild-type gene fragment, analyzing for the presence of a mismatch in theheteroduplex, and sequencing the first strand of nucleic acid having amismatch; (j) forming single-stranded DNA from the gene of the humansample and from a corresponding fragment of an allele specific for oneof the alterations, electrophoresing the single-stranded DNAs on anon-denaturing polyacrylamide gel and comparing the mobility of thesingle-stranded DNAs on the gel to determine if the single-stranded DNAfrom the sample is shifted relative to the allele, wherein no shift inelectrophoretic mobility of the single-stranded DNA relative to theallele indicates the presence of the alteration in the sample; and (k)forming a heteroduplex consisting of a first strand of nucleic acidselected from the group consisting of a genomic DNA fragment of the geneisolated from the sample, an RNA fragment isolated from the sample and acDNA fragment made from mRNA from the sample and a second strand of anucleic acid consisting of a corresponding gene allele fragment specificfor one of the alterations and analyzing for the presence of a mismatchin the heteroduplex, wherein no mismatch indicates the presence of thealteration.

In an eleventh aspect, the invention provides a method for determiningwhether a human subject has or is at risk for developing depression. Inone embodiment, the method involves: (a) obtaining a sample from asubject, said sample comprising nucleic acid molecules containing APAF1gene; and (b) detecting the presence or absence of a genetic alterationin the gene of said subject, wherein the presence of said geneticalteration identifies a subject that has or is at risk for developingdepression. In another embodiment, the alteration (mutation) is selectedfrom the group consisting of C1350G, A1394G, G2329A, A2345C, A2857G,insertion of a T after nucleotide position 1299, T1244C, C1070T, C1437G,A1874C or complements thereof (referring to SEQ ID NO: 1).

In a twelfth aspect, the invention provides a method for preventing ortreating depression in a subject which comprises administering to thesubject a therapeutically effective amount of a compound that agonizesor antagonizes wild-type APAF1, or agonizes or antagonizes alteredAPAF1, or agonizes or antagonizes an APAF1 receptor. In one embodiment,the method for preventing or treating depression involves a compound isselected from: (a) a drug; (b) an antisense molecule; (c) an siRNAmolecule; (d) a ribozyme; (e) a triplex molecule; (f) wild-type APAF1nucleic acid; (g) wild-type APAF1 protein; (h) a protein which bindswild-type or altered APAF1 protein; (i) a peptidomimetic; (j) anon-peptide, non-nucleic acid small molecule; and (k) an antibody.

In a thirteenth aspect, the invention provides a non-human animal whichcarries an altered APAF1 allele in its genome. According to oneembodiment of this aspect of the invention, a cell line isolated fromthe non-human animal is provided.

In a fourteenth aspect, the invention provides a method of screening fordrug candidates useful in treating depression resulting from analteration in APAF1. The method, according to one embodiment, involvesmixing a mutant APAF1 in both the presence of a drug and the absence ofthe drug and measuring the level of the biological activity of themutant APAF1. If the level of the biological activity is less in thepresence of the drug than in the absence of the drug then it is a drugcandidate for treating depression. According to another embodiment ofthis aspect of the invention, the method involves treating an animal,which is heterozygous or homozygous for APAF1 containing an alteration,with a drug. If the animal does not develop depression, or symptomsthereof, then the drug is a candidate for treating depression. In yetanother embodiment of this aspect of the invention, a method forscreening potential depression therapeutics is provided. The methodaccording to this embodiment involves combining an APAF1 binding partnerand a compound suspected of being a depression therapeutic and measuringthe biological activity of the binding partner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a SDS-PAGE gel demonstrating that APAF1 mutants segregatingwith depression are capable of reconstituting apoptosome activity. SeeExample 2 for experimental details. Experiment performed at 0.3 mM[caspase], 0.3 or 0.6 mM [Apaf-1], 0.3 mM cytochrome C. Conversion ofprocaspase-9 to caspase 9 is clearly evident as indicated by the bandsat the arrows.

FIG. 2 shows a western blot demonstrating that APAF1 mutants segregatingwith depression are capable of reconstituting apoptosome activity. SeeExample 2 for experimental details. Experiment performed at 0.3 mM[caspase], 0.3 or 0.6 mM [Apaf-1], 0.3 mM cytochrome C. Conversion ofprocaspase-9 to caspase-9 is clearly evident as indicated by the bandsat the arrows.

FIG. 3 is a bar graph showing the affect of APAF1 mutants identified asbeing associated with depression on caspase activation in an in vitroapoptosome assay. See Example 2 for experimental details. FIG. 3indicates that mutations in Apaf-1 that segregate with depression resultin increased activation of caspase-9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery of polymorphismsin the APAF1 gene which are linked to depression. Five differentmissense changes were discovered in linked families in this gene whichresult in a Cys->Trp substitution at amino acid 450 (C450W) in family8546 (nucleotide change c1350g), a Gln->Arg substitution at amino acid465 (Q465R) in family 8428 (nucleotide change a1394g), a Glu->Lyssubstitution at amino acid 777 (E777K) in family 8347.2 (nucleotidechange g2329a), an Asn->Thr substitution at amino acid 782 (A782T) infamily 8288 (nucleotide change a2345c) and a Thr->Ala substitution atamino acid 953 (T953A) in family 8828803 (nucleotide change a2857g).Further analysis of APAF1 has uncovered three other mutationscorresponding to Ser357Leu; Asp479Glu; and Glu625Ala with respectivenucleotide changes are C1070T; C1437G; and A1874C. Each one occurs on ahaplotype that segregates into more than one affected individual.Additional 180 male affected cases were examined. To date one frameshiftmutation 1299insT (inserts stop codon at codon 439) in family 8205 andanother missense change Leu->Pro at amino acid 415 (L415P) in family8582 (nucleotide change t1244c), were discovered in this random caseset. Neither of these changes were seen in 177 control samples. Theprotein encoded by the frameshift mutation is set forth in SEQ ID NO:3.Based on these findings, the invention provides therapeutic methods,compositions and diagnostics for depression based on APAF1.

The cDNA for the APAF1 gene and the protein sequence are set forth inGenBank accession number AF149794. The coding sequence for APAF1 as usedherein is shown in SEQ ID NO:1. The corresponding amino acid sequencefor APAF1 is set forth in SEQ ID NO:2. The present invention relates toAPAF1 agonists and antagonists and their use in treating depression. Forexample, (i) nucleic acid molecules encoding functional APAF1 protein;(ii) nucleic acids that are effective antisense, siRNA, ribozyme andtriplex antagonists of nucleic acids encoding functional APAF1 protein;(iii) functional APAF1 proteins or peptides; (iv) anti-APAF1 antibodies;(v) drugs affecting wild-type or mutant APAF1 function or APAF1interaction with an APAF1 receptor (or interacting partner) andpreparations of such compositions are disclosed herein. In addition, theinvention provides drug discovery assays for identifying additionalagents that agonize or antagonize the biological function of APAF1protein (e.g. by altering the interaction of APAF1 molecules with eitherdownstream or upstream elements in the biochemical (e.g. signaltransduction) pathway). Moreover, the present invention provides assaysfor diagnosing whether a subject has depression or has a predispositiontowards developing depression.

Proof that any particular gene located within the genetically definedinterval is a disease susceptibility locus is obtained by findingsequences in DNA or RNA extracted from affected kindred members whichcreate abnormal gene products or abnormal levels of gene product. Suchdisease susceptibility alleles will co-segregate with the disease inlarge kindreds. They will also be present at a much higher frequency innon-kindred individuals with the disease than in individuals in thegeneral population. In identifying a disease susceptibility locus, thekey is to find polymorphisms or mutations which are serious enough tocause obvious disruption to the normal function of the gene product.These mutations can take a number of forms. The most severe forms aretypically frame shift mutations or large deletions which cause the geneto code for an abnormal protein or one which significantly altersprotein expression. Less severe disruptive mutations include smallin-frame deletions and non-conservative base pair substitutions whichhave a significant effect on the protein produced, such as changes to orfrom a cysteine residue, from a basic to an acidic amino acid or viceversa, from a hydrophobic to hydrophilic amino acid or vice versa, orother mutations which affect secondary, tertiary or quaternary proteinstructure. Small deletions or base pair substitutions can alsosignificantly alter protein expression by changing the level oftranscription, splice pattern, mRNA stability, or translation efficiencyof the gene transcript. Silent mutations or those resulting inconservative amino acid substitutions are not generally expected todisrupt protein function. Causal mutations can also be found in thepromoter of the gene. These mutations interfere with the binding ofregulatory factors and in this way alter transcription of the gene andtherefore change the function of the gene. Since the inventors havediscovered that APAF1 mutations segregate with major depression,Applicants contemplate that the abovementioned classes of mutations canoccur in APAF1 and be associated with depression.

Diagnostics

The discovery by the present inventors that APAF1 mutations segregatewith depression now allows for depression susceptibility testing basedon detecting mutations in APAF1.

In one aspect, the invention features probes and primers for use in aprognostic or diagnostic assay. For instance, the present invention alsoprovides a probe/primer comprising a substantially purifiedoligonucleotide, which oligonucleotide comprises a region of nucleotidesequence that hybridizes under stringent conditions to at leastapproximately 12, preferably 25, more preferably 40, 50 or 75consecutive nucleotides of sense or antisense sequence of APAF1,including 5′ and/or 3′ untranslated regions. In preferred embodiments,the probe further comprises a detectable label group attached thereto,e.g. the label group is selected from amongst radioisotopes, fluorescentcompounds, enzymes, and enzyme co-factors. The selection of probes andprimers for diagnosis of a susceptibility of depression (i.e, detectionof APAF1 mutations) is within the capability of the skilled artisanapprised of the invention.

In a further aspect, the present invention features methods fordetermining whether a subject is at risk for developing depression.According to the diagnostic and prognostic methods of the presentinvention, alteration of the wild-type APAF1 locus is detected.“Alteration of a wild-type gene” encompasses all forms of mutationsincluding deletions, insertions and point mutations in the coding andnon-coding regions. Deletions may be of the entire gene or of only aportion of the gene. Point mutations may result in stop codons,frameshift mutations or amino acid substitutions. Point mutations ordeletions in the promoter can change transcription and thereby alter thegene function. Somatic mutations are those which occur only in certaintissues and are not inherited in the germline. Germline mutations can befound in any of a body's tissues and are inherited. The finding of APAF1germline mutations thus provides diagnostic information. An APAF1 allelewhich is not deleted (e.g., found on the sister chromosome to achromosome carrying an APAF1 deletion) can be screened for othermutations, such as insertions, small deletions, and point mutations.Point mutational events may occur in regulatory regions, such as in thepromoter of the gene, or in intron regions or at intron/exon junctions.

Useful diagnostic techniques include, but are not limited to fluorescentin situ hybridization (FISH), direct DNA sequencing, PFGE (pulsed-fieldgel electrophoresis) analysis, Southern blot analysis, single strandedconformation analysis (SSCA), RNase protection assay, allele-specificoligonucleotide (ASO), dot blot analysis and PCR-SSCP, as discussed indetail further below. Also useful is the recently developed technique ofDNA microchip technology. In addition to the techniques describedherein, similar and other useful techniques are also described in U.S.Pat. Nos. 5,837,492 and 5,800,998, each incorporated herein byreference.

Predisposition to disease can be ascertained by testing any tissue of ahuman for mutations of the APAF1 gene. For example, a person who hasinherited a germline APAF1 mutation may be prone to developingdepression. This can be determined by testing DNA from any tissue of theperson's body. Most simply, blood can be drawn and DNA extracted fromthe cells of the blood. In addition, prenatal diagnosis can beaccomplished by testing fetal cells, placental cells or amniotic cellsfor mutations of the APAF1 gene. Alteration of a wild-type APAF1 allele,whether, for example, by point mutation or deletion, can be detected byany of the means discussed herein.

There are several methods that can be used to detect DNA sequencevariation. Direct DNA sequencing, either manual sequencing or automatedfluorescent sequencing can detect sequence variation. Another approachis the single-stranded conformation polymorphism assay (SSCA) (Orita, etal. (1989) Proc. Natl. Acad. Sci. USA 86:2776 2770). This method doesnot detect all sequence changes, especially if the DNA fragment size isgreater than 200 bp, but can be optimized to detect most DNA sequencevariation. The reduced detection sensitivity is a disadvantage, but theincreased throughput possible with SSCA makes it an attractive, viablealternative to direct sequencing for mutation detection on a researchbasis. The fragments which have shifted mobility on SSCA gels are thensequenced to determine the exact nature of the DNA sequence variation.Other approaches based on the detection of mismatches between the twocomplementary DNA strands include clamped denaturing gel electrophoresis(CDGE) (Sheffield, V. C., et al. (1991) Am. J. Hum. Genet. 49:699 706),heteroduplex analysis (HA) (White, M. B., et al., (1992) Genomics 12:301306) and chemical mismatch cleavage (CMC) (Grompe, M., et al., (1989)Proc. Natl. Acad. Sci. USA 86:5855 5892). None of the methods describedabove will detect large deletions, duplications or insertions, nor willthey detect a regulatory mutation which affects transcription ortranslation of the protein. Other methods which can detect these classesof mutations such as a protein truncation assay or the asymmetric assay,detect only specific types of mutations and would not detect missensemutations. A review of currently available methods of detecting DNAsequence variation can be found in a recent review by Grompe, M., (1993)Nature Genetics 5:111 117. Once a mutation is known, an allele specificdetection approach such as allele specific oligonucleotide (ASO)hybridization can be utilized to rapidly screen large numbers of othersamples for that same mutation.

Detection of point mutations may be accomplished by molecular cloning ofthe APAF1 allele(s) and sequencing the allele(s) using techniques wellknown in the art. Alternatively, the gene sequences can be amplifieddirectly from a genomic DNA preparation from the tissue or cells, usingknown techniques. The DNA sequence of the amplified sequences can thenbe determined.

There are six well known methods for a more complete, yet stillindirect, test for confirming the presence of a susceptibilityallele: 1) single-stranded conformation analysis (SSCA) (Orita, et al.(1989) Proc. Natl. Acad. Sci. USA 86:2776 2770); 2) denaturing gradientgel electrophoresis (DGGE) (Wartell, R. M., et al. (1990) Nucl. AcidsRes. 18:2699 2705; Sheffield, V. C., et al. (1989) Proc. Natl. Acad.Sci. USA 86:232 236); 3) RNase protection assays (Finkelstein, J., etal. (1990) Genomics 7:167 172; Kinszler, K. W., et al. (1991) Science251:1366 1370); 4) allele-specific oligonucleotides (ASOs) (Conner, B.J., et al. (1983) Proc. Natl. Acad. Sci. USA 80:278 282); 5) the use ofproteins which recognize nucleotide mismatches, such as the E. coli mutSprotein (Modrich, P. (1991) Ann. Rev. Genet. 25:229 253); and 6)allele-specific PCR (Rano & Kidd (1989) Nucl. Acids Res. 17:8392). Forallele-specific PCR, primers are used which hybridize at their 3′ endsto a particular APAF1 mutation. If the particular APAF1 mutation is notpresent, an amplification product is not observed. AmplificationRefractory Mutation System (ARMS) can also be used, as disclosed inEuropean Patent Application Publication No. 0332435 and in Newton, C.R., et al. (1989) Nucl. Acids Res. 17:2503 2516. Insertions anddeletions of genes can also be detected by cloning, sequencing andamplification. In addition, restriction fragment length polymorphism(RFLP) probes for the gene or surrounding marker genes can be used toscore alteration of an allele or an insertion in a polymorphic fragment.Such a method is particularly useful for screening relatives of anaffected individual for the presence of the APAF1 mutation found in thatindividual. Other techniques for detecting insertions and deletions asknown in the art can be used.

In the first three methods (SSCA, DGGE and RNase protection assay), anew electrophoretic band appears. SSCA detects a band which migratesdifferentially because the sequence change causes a difference insingle-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild-typesequences.

Mismatches, according to the present invention, are hybridized nucleicacid duplexes in which the two strands are not 100% complementary. Lackof total homology may be due to deletions, insertions, inversions orsubstitutions. Mismatch detection can be used to detect point mutationsin the gene or in its mRNA product. While these techniques are lesssensitive than sequencing, they are simpler to perform on a large numberof tumor samples. An example of a mismatch cleavage technique is theRNase protection method. In the practice of the present invention, themethod involves the use of a labeled riboprobe which is complementary tothe human wild-type APAF1 gene coding sequence. The riboprobe and eithermRNA or DNA isolated from the tumor tissue are annealed (hybridized)together and subsequently digested with the enzyme RNase A which is ableto detect some mismatches in a duplex RNA structure. If a mismatch isdetected by RNase A, it cleaves at the site of the mismatch. Thus, whenthe annealed RNA preparation is separated on an electrophoretic gelmatrix, if a mismatch has been detected and cleaved by RNase A, an RNAproduct will be seen which is smaller than the full length duplex RNAfor the riboprobe and the mRNA or DNA. The riboprobe need not be thefull length of the APAF1 mRNA or gene but can be a segment of either. Ifthe riboprobe comprises only a segment of the APAF1 mRNA or gene, itwill be desirable to use a number of these probes to screen the wholemRNA sequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton, et al. (1988) Proc.Natl. Acad. Sci. USA 85:4397 4401; Shenk, et al. (1975) Proc. Natl.Acad. Sci. USA 72:989; and Novack, et al. (1986) Proc. Natl. Acad. Sci.USA 83:586. Alternatively, mismatches can be detected by shifts in theelectrophoretic mobility of mismatched duplexes relative to matchedduplexes. See, e.g., Cariello (1988) Human Genetics 42:726. With eitherriboprobes or DNA probes, the cellular mRNA or DNA which might contain amutation can be amplified using PCR before hybridization. Changes in DNAof the APAF1 gene can also be detected using Southern hybridization,especially if the changes are gross rearrangements, such as deletionsand insertions.

DNA sequences of the APAF1 gene which have been amplified by use of PCRmay also be screened using allele-specific probes. These probes arenucleic acid oligomers, each of which contains a region of the APAF1gene sequence harboring a known mutation. For example, one oligomer maybe about 30 nucleotides in length (although shorter and longer oligomersare also usable as well recognized by those of skill in the art),corresponding to a portion of the APAF1 gene sequence. By use of abattery of such allele-specific probes, PCR amplification products canbe screened to identify the presence of a previously identified mutationin the APAF1 gene. Hybridization of allele-specific probes withamplified APAF1 sequences can be performed, for example, on a nylonfilter. Hybridization to a particular probe under high stringencyhybridization conditions indicates the presence of the same mutation inthe tumor tissue as in the allele-specific probe.

The newly developed technique of nucleic acid analysis via microchiptechnology is also applicable to the present invention. In thistechnique, literally thousands of distinct oligonucleotide probes arebuilt up in an array on a silicon chip. Nucleic acid to be analyzed isfluorescently labeled and hybridized to the probes on the chip. It isalso possible to study nucleic acid-protein interactions using thesenucleic acid microchips. Using this technique one can determine thepresence of mutations, sequence the nucleic acid being analyzed, and/orone can measure expression levels of a gene of interest. The method isone of parallel processing of many, even thousands, of probes at onceand can tremendously increase the rate of analysis. Several papers havebeen published which use this technique. See, e.g., Hacia J G, et al.(1996) Nature Genetics 14:441 447; Shoemaker D D, et al. (1996) NatureGenetics 14:450 456; Chee, M., et al. (1996) Science 274:610 614;Lockhart D J, et al. (1996) Nature Biotechnology 14:1675 1680; DeRisi,J., et al. (1996) Nat. Genet. 14:457 460; Lipshutz R J, et al. (1995)BioTechniques 19:442 447. This method has already been used to screenpeople for mutations in the breast cancer gene BRCA1 (Hacia, J G, et al.(1996) Nature Genetics 14:441 447). This new technology has beenreviewed in a news article in Chemical and Engineering News (Borman, S.(1996) Chemical & Engineering News, December 9 issue, pp. 42 43) andbeen the subject of an editorial (Nature Genetics, 1996). Also seeFodor, S. P. A. (1997) Science 277:393 395.

The most definitive test for mutations in a candidate locus is todirectly compare genomic APAF1 sequences from disease patients withthose from a control population. Alternatively, one could sequencemessenger RNA after amplification, e.g., by PCR, thereby eliminating thenecessity of determining the exon structure of the candidate gene.

Mutations from disease patients falling outside the coding region ofAPAF1 can be detected by examining the non-coding regions, such asintrons and regulatory sequences near or within the APAF1 gene. An earlyindication that mutations in non-coding regions are important can comefrom Northern blot experiments that reveal messenger RNA molecules ofabnormal size or abundance in disease patients as compared to controlindividuals.

Alteration of APAF1 mRNA expression can be detected by any techniquesknown in the art. These include Northern blot analysis, PCRamplification and RNase protection. Diminished or increased mRNAexpression indicates an alteration of the wild-type APAF1 gene.Alteration of wild-type APAF1 genes can also be detected by screeningfor alteration of wild-type APAF1 protein. For example, monoclonalantibodies immunoreactive with APAF1 can be used to screen a tissue.Lack of cognate antigen would indicate an APAF1 mutation. Antibodiesspecific for products of mutant alleles could also be used to detectmutant APAF1 gene product. Such immunological assays can be done in anyconvenient formats known in the art. These include Western blots,immunohistochemical assays and ELISA assays. Any means for detecting analtered APAF1 protein can be used to detect alteration of wild-typeAPAF1 genes. Functional assays, such as protein binding determinations,can be used. In addition, assays can be used which detect APAF1biochemical function. Finding a mutant APAF1 gene product indicatesalteration of a wild-type APAF1 gene.

The primer pairs of the present invention are useful for determinationof the nucleotide sequence of a particular APAF1 allele using PCR. Thepairs of single-stranded DNA primers can be annealed to sequences withinor surrounding the APAF1 gene on chromosome 12 in order to primeamplifying DNA synthesis of the APAF1 gene itself. A complete set ofthese primers allows synthesis of all of the nucleotides of the APAF1gene coding sequences, i.e., the exons. The set of primers preferablyallows synthesis of both intron and exon sequences. Allele-specificprimers can also be used. Such primers anneal only to particular APAF1mutant alleles, and thus will only amplify a product in the presence ofthe mutant allele as a template.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme site sequences appended to their 5′ends. Thus, all nucleotides of the primers are derived from APAF1sequences or sequences adjacent to APAF1, except for the few nucleotidesnecessary to form a restriction enzyme site. Such enzymes and sites arewell known in the art. The primers themselves can be synthesized usingtechniques which are well known in the art. Generally, the primers canbe made using oligonucleotide synthesizing machines which arecommercially available. Given the known sequences of the APAF1 exons andthe 5′ alternate exon, the design of particular primers is well withinthe skill of the art. Suitable primers for mutation screening are alsodescribed herein.

The nucleic acid probes provided by the present invention are useful fora number of purposes. They can be used in Southern hybridization togenomic DNA and in the RNase protection method for detecting pointmutations already discussed above. The probes can be used to detect PCRamplification products. They can also be used to detect mismatches withthe APAF1 gene or mRNA using other techniques.

It has been discovered that individuals with the wild-type APAF1 gene donot have depression which results from the APAF1 allele. However,mutations which interfere with the function of the APAF1 protein areinvolved in the susceptibility to depression as shown herein. Thus, thepresence of an altered (or a mutant) APAF1 gene which produces a proteinhaving a loss of function, or altered function, directly correlates toan increased risk of disease. In order to detect an APAF1 gene mutation,a biological sample is prepared and analyzed for a difference betweenthe sequence of the APAF1 allele being analyzed and the sequence of thewild-type APAF1 allele. Mutant APAF1 alleles can be initially identifiedby any of the techniques described above. The mutant alleles are thensequenced to identify the specific mutation of the particular mutantallele. Alternatively, mutant APAF1 alleles can be initially identifiedby identifying mutant (altered) APAF1 proteins, using conventionaltechniques. The mutant alleles are then sequenced to identify thespecific mutation for each allele. The mutations, especially those whichlead to an altered function of the APAF1 protein, are then used for thediagnostic methods of the present invention.

The present invention employs definitions commonly used in the art withspecific reference to the gene described in the present application.Such definitions can be found in U.S. Pat. Nos. 5,837,492; 5,800,998;6,261,801; 6,274,720 and 6,274,376, each incorporated herein byreference. Such definitions are employed herein unless the contextindicates otherwise.

Nucleic Acids and Proteins

A nucleic acid or fragment thereof has substantial identity with anotherif, when optimally aligned (with appropriate nucleotide insertions ordeletions) with the other nucleic acid (or its complementary strand),there is nucleotide sequence identity in at least about 60% of thenucleotide bases, usually at least about 70%, more usually at leastabout 80%, preferably at least about 90%, and more preferably at leastabout 95 98% of the nucleotide bases. A protein or fragment thereof hassubstantial identity with another if, optimally aligned, there is anamino acid sequence identity of at least about 30% identity with anentire naturally-occurring protein or a portion thereof, usually atleast about 70% identity, more usually at least about 80% identity,preferably at least about 90% identity, and more preferably at leastabout 95% identity.

Identity means the degree of sequence relatedness between twopolypeptide or two polynucleotide sequences as determined by theidentity of the match between two strings of such sequences, such as thefull and complete sequence. Identity can be readily calculated. Whilethere exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). Methods commonly employed to determine identity betweentwo sequences include, but are not limited to, those disclosed in Guideto Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego,1994, and Carillo, H., and Lipman, D., SIAM J Applied Math. 48:1073(1988). Preferred methods to determine identity are designed to give thelargest match between the two sequences tested. Such methods arecodified in computer programs. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, GCG (Genetics Computer Group, Madison Wis.) program package(Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP,BLASTN, FASTA (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410;and Altschul, S. F. et al. (1997) Nucl. Acids Res. 25:3389 3402). Thewell-known Smith Waterman algorithm may also be used to determineidentity.

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence. In other words, toobtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence can occur at the 5′or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

Alternatively, substantial homology or (similarity) exists when anucleic acid or fragment thereof will hybridize to another nucleic acid(or a complementary strand thereof) under selective hybridizationconditions, to a strand, or to its complement. Selectivity ofhybridization exists when hybridization which is substantially moreselective than total lack of specificity occurs. Typically, selectivehybridization occurs when there is at least about 55% homology over astretch of at least about 14 nucleotides, preferably at least about 65%,more preferably at least about 75%, and most preferably at least about90%. The length of homology comparison, as described, can be over longerstretches, and in certain embodiments can be over a stretch of at leastabout nine nucleotides, usually at least about 20 nucleotides, moreusually at least about 24 nucleotides, typically at least about 28nucleotides, more typically at least about 32 nucleotides, andpreferably at least about 36 or more nucleotides.

Nucleic acid hybridization can be affected by such conditions as saltconcentration, temperature, or organic solvents, in addition to the basecomposition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, as isreadily appreciated by those skilled in the art. Stringent temperatureconditions will generally include temperatures in excess of 30° C.,typically in excess of 37° C., and preferably in excess of 45° C.Stringent salt conditions will ordinarily be less than 1000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. The stringency conditions are dependent on thelength of the nucleic acid and the base composition of the nucleic acid,and can be determined by techniques well known in the art. See, e.g.,Ausubel, F. M., et al. (1992) Current Protocols in Molecular Biology,(J. Wiley and Sons, NY); Wetmur, J. G. and Davidson, N. (1968) J. Mol.Biol. 31:349 370.

Thus, as herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences. Such hybridization techniquesare well known to those of skill in the art. Stringent hybridizationconditions are as defined above or, alternatively, conditions underovernight incubation at 42° C. in a solution comprising: 50% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate(pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65° C.

The terms “isolated”, “substantially pure”, and “substantiallyhomogeneous” are used interchangeably to describe a protein orpolypeptide which has been separated from components which accompany itin its natural state. A monomeric protein is substantially pure when atleast about 60 to 75% of a sample exhibits a single polypeptidesequence. A substantially pure protein can typically comprise about 60to 90% W/W of a protein sample, more usually about 95%, and preferablycan be over about 99% pure. Protein purity or homogeneity can beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band upon staining the gel. For certainpurposes, higher resolution can be provided by using HPLC or other meanswell known in the art which are utilized for purification.

Large amounts of the nucleic acids of the present invention can beproduced by (a) replication in a suitable host or transgenic animals or(b) chemical synthesis using techniques well known in the art.Constructs prepared for introduction into a prokaryotic or eukaryotichost may comprise a replication system recognized by the host, includingthe intended polynucleotide fragment encoding the desired polypeptide,and will preferably also include transcription and translationalinitiation regulatory sequences operably linked to the polypeptideencoding segment. Expression vectors include, for example, an origin ofreplication or autonomously replicating sequence (ARS) and expressioncontrol sequences, a promoter, an enhancer and necessary processinginformation sites, such as ribosome-binding sites, RNA splice sites,polyadenylation sites, transcriptional terminator sequences, and mRNAstabilizing sequences. Secretion signals can also be included whereappropriate which allow the protein to cross and/or lodge in cellmembranes, and thus attain its functional topology, or be secreted fromthe cell. Such vectors may be prepared by means of standard recombinanttechniques well known in the art.

Methods of Use: Nucleic Acid Diagnosis and Diagnostic Kits

In order to detect the presence of an APAF1 allele predisposing anindividual to depression, a biological sample such as blood is preparedand analyzed for the presence or absence of predisposing alleles ofAPAF1. Results of these tests and interpretive information are returnedto the health care provider for communication to the tested individual.Such diagnoses can be performed by diagnostic laboratories, or,alternatively, diagnostic kits are manufactured and sold to health careproviders or to private individuals for self-diagnosis. Diagnositic orprognostic tests can be performed as described herein or using wellknown techniques, such as described in U.S. Pat. No. 5,800,998,incorporated herein by reference.

Initially, the screening method can involve amplification of therelevant APAF1 sequences. In another preferred embodiment of theinvention, the screening method involves a non-PCR based strategy. Suchscreening methods include two-step label amplification methodologiesthat are well known in the art. Both PCR and non-PCR based screeningstrategies can detect target sequences with a high level of sensitivity.

The most popular method used today is target amplification. Here, thetarget nucleic acid sequence is amplified with a polymerase. Oneparticularly preferred method using polymerase-driven amplification isthe polymerase chain reaction (PCR). The polymerase chain reaction andother polymerase-driven amplification assays can achieve over amillion-fold increase in copy number through the use ofpolymerase-driven amplification cycles. Once amplified, the resultingnucleic acid can be sequenced or used as a substrate for DNA probes.

When the probes are used to detect the presence of the target sequences(for example, in screening for depression susceptibility), thebiological sample to be analyzed, such as blood or serum, may betreated, if desired, to extract the nucleic acids. The sample nucleicacid can be prepared in various ways to facilitate detection of thetarget sequence; e.g. denaturation, restriction digestion,electrophoresis or dot blotting. The targeted region of the analytenucleic acid usually must be at least partially single-stranded to formhybrids with the targeting sequence of the probe. If the sequence isnaturally single-stranded, denaturation will not be required. However,if the sequence is double-stranded, the sequence will probably need tobe denatured. Denaturation can be carried out by various techniquesknown in the art.

Analyte nucleic acid and probe are incubated under conditions whichpromote stable hybrid formation of the target sequence in the probe withthe putative targeted sequence in the analyte. The region of the probeswhich is used to bind to the analyte can be made completelycomplementary to the targeted region of human chromosome 12. Therefore,high stringency conditions are desirable in order to prevent falsepositives. However, conditions of high stringency are used only if theprobes are complementary to regions of the chromosome which are uniquein the genome. The stringency of hybridization is determined by a numberof factors during hybridization and during the washing procedure,including temperature, ionic strength, base composition, probe length,and concentration of formamide. These factors are outlined in, forexample, Maniatis T., et al. (1982) Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) andSambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, 2ndEd. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Undercertain circumstances, the formation of higher order hybrids, such astriplexes, quadraplexes, etc., may be desired to provide the means ofdetecting target sequences.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labeled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand which islabeled, either directly or indirectly. Suitable labels, and methods forlabeling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labeledmoiety. A number of these variations are reviewed in, e.g., Matthews andKricka (1988) Anal. Biochem. 169:1; Landegren, et al. (1988) Science242:229; Mittlin (1989) Clinical Chem. 35:1819; U.S. Pat. No. 4,868,105,and in EPO Publication No. 225,807.

As noted above, non-PCR based screening assays are also contemplated inthis invention. This procedure hybridizes a nucleic acid probe (or ananalog such as a methyl phosphonate backbone replacing the normalphosphodiester), to the low level DNA target. This probe can have anenzyme covalently linked to the probe, such that the covalent linkagedoes not interfere with the specificity of the hybridization. Thisenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe enzyme conjugate and a substrate is added forenzyme detection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in a 10³ 10⁶ increase insensitivity. For an example relating to the preparation ofoligodeoxynucleotide-alkaline phosphatase conjugates and their use ashybridization probes see Jablonski, E., et al. (1986) Nuc. Acids Res.14:6115 6128.

Two-step label amplification methodologies are known in the art. Theseassays work on the principle that a small ligand (such as digoxigenin,biotin, or the like) is attached to a nucleic acid probe capable ofspecifically binding APAF1. Allele specific probes are also contemplatedwithin the scope of this example and exemplary allele specific probesinclude probes encompassing the predisposing or potentially predisposingmutations summarized in herein.

In one example, the small ligand attached to the nucleic acid probe isspecifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digoxigenin is attached to the nucleic acidprobe. Hybridization is detected by an antibody-alkaline phosphataseconjugate which turns over a chemiluminescent substrate. For methods forlabeling nucleic acid probes according to this embodiment see Martin etal., 1990. In a second example, the small ligand is recognized by asecond ligand-enzyme conjugate that is capable of specificallycomplexing to the first ligand. A well known embodiment of this exampleis the biotin-avidin type of interactions. For methods for labelingnucleic acid probes and their use in biotin-avidin based assays seeRigby, P. W. J., et al. (1977) J. Mol. Biol. 113:237 251 and Nguyen, Q.,et al. (1992) BioTechniques 13:116 123.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention will employ a cocktail ofnucleic acid probes capable of detecting APAF1. Thus, in one example todetect the presence of APAF1 in a cell sample, more than one probecomplementary to APAF1 is employed and in particular the number ofdifferent probes is alternatively 2, 3, or 5 different nucleic acidprobe sequences. In another example, to detect the presence of mutationsin the APAF1 gene sequence in a patient, more than one probecomplementary to APAF1 is employed where the cocktail includes probescapable of binding to the allele-specific mutations identified inpopulations of patients with alterations in APAF1. In this embodiment,any number of probes can be used, and can preferably include probescorresponding to the major gene mutations identified as predisposing anindividual to depression. Some candidate probes contemplated within thescope of the invention include probes that include the allele-specificmutations identified herein and those that have the APAF1 regionscorresponding to SEQ ID NOs:1 5 and 8 both 5′ and 3′ to the mutationsite.

Methods of Use: Peptide Diagnosis and Diagnostic Kits

Susceptibility to depression can also be detected on the basis of thealteration of wild-type APAF1 polypeptide. Peptide diagnostic orprognostic tests can be performed as described herein or using wellknown techniques, such as described in U.S. Pat. No. 5,800,998,incorporated herein by reference. For example, such alterations can bedetermined by sequence analysis in accordance with conventionaltechniques. More preferably, antibodies (polyclonal or monoclonal) areused to detect differences in, or the absence of, APAF1 peptides. Theantibodies can be prepared in accordance with conventional techniques.Other techniques for raising and purifying antibodies are well known inthe art and any such techniques may be chosen to achieve thepreparations claimed in this invention. In a preferred embodiment of theinvention, antibodies will immunoprecipitate APAF1 proteins or fragmentsof the APAF1 protein from solution as well as react with APAF1 peptideson Western or immunoblots of polyacrylamide gels. In another preferredembodiment, antibodies will detect APAF1 proteins and protein fragmentsin paraffin or frozen tissue sections, using immunocytochemicaltechniques.

Preferred embodiments relating to methods for detecting APAF1 (ormutants thereof) include enzyme linked immunosorbent assays (ELISA),radioimmunoassays (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal and/or polyclonal antibodies. Exemplary sandwich assays aredescribed by David et al. in U.S. Pat. Nos. 4,376,110 and 4,486,530,hereby incorporated by reference.

Methods of Use: Drug Screening

Polypeptides of the invention also may be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2):Chapter 5 (1991). Thus, the invention alsoprovides a method of screening compounds to identify those which enhance(agonist) or block (antagonist) the action of APAF1 polypeptides orpolynucleotides, particularly those compounds for treating or preventingdepression.

This invention is particularly useful for screening compounds by using awild-type or mutant APAF1 polypeptide or a binding fragment thereof inany of a variety of drug screening techniques. The individual componentsof the assays described herein are available commercially and/or can beproduced by an ordinary skilled artisan. APAF1 is described in U.S. Pat.No. 6,346,607 to Wang, issued Feb. 12, 2002, which is hereinincorporated by reference in its entirety. The screens of the inventionare intended to encompass the use of APAF1 homologs and APAF1interacting proteins from any organism including, C. elegans andDrosphila, as well as human. The skilled artisan is capable ofrecognizing and employing APAF1 homologs and homologs of APAF1interacting partners from other organisms in the assays of theinvention. Drug screening can be performed as described herein or usingwell known techniques, such as described in U.S. Pat. Nos. 5,800,998 and5,891,628, each incorporated herein by reference. Preferably testcompounds that disrupt APAF1 bioactivity are tested in a secondary assaysuch as an animal depression model, a cellular based apoptosis assay,and transgenic animal models being homozygous or heterozygous for anAPAF1 mutation that is associated with depression. Furthermore, as theskilled artisan readily understands, the screening assays describedherein can be performed in a variety of configurations based on knownAPAF1 biochemistry, including, but not limited to testing to disrupt ofinhibit apoptosome formation, inhibiting binding of ATP, dATP or anappropriate analog thereof from binding APAF1 and facilitatingapoptosome formation, disruption or inhibition of cytochrome c bindingto APAF1, enhancement of the WD40 repeates of APAF1 to autoinhibit theformation of apoptosome formation, and inhibition or disruption ofprocaspase-9 binding to the CARD (caspase recruitment domain) of APAF1.The assays can readily be configured to use a full apoptosomereconstitution assay or they can be based on inhibiting, e.g.,protein:protein interactions, between members of the apoptosome usingfragments of the proteins or full length proteins where appropriate. Apreferred drug screen involves the use of an apoptosome reconstitutionassay as described herein to identify compounds that inhibit or reducecaspase activation. A few representative embodiments are describedbelow.

The APAF1 polypeptide or fragment employed in such a test can either befree in solution, affixed to a solid support, or borne on a cellsurface. One method of drug screening utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant polynucleotidesexpressing the polypeptide or fragment, preferably in competitivebinding assays. Such cells, either in viable or fixed form, can be usedfor standard binding assays. One may measure, for example, for theformation of complexes between an APAF1 polypeptide or fragment and theagent being tested, or examine the degree to which the formation of acomplex between an APAF1 polypeptide or fragment and a known ligand,e.g. APAF1 receptor (AT1), is interfered with by the agent being tested.

Thus, the present invention provides methods of screening for drugscomprising contacting such an agent with an APAF1 polypeptide orfragment thereof and assaying (i) for the presence of a complex betweenthe agent and the APAF1 polypeptide or fragment, or (ii) for thepresence of a complex between the APAF1 polypeptide or fragment and aligand, by methods well known in the art. In such competitive bindingassays the APAF1 polypeptide or fragment is typically labeled. FreeAPAF1 polypeptide or fragment is separated from that present in aprotein:protein complex, and the amount of free (i.e., uncomplexed)label is a measure of the binding of the agent being tested to APAF1 orits interference with APAF1:ligand binding, respectively.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the APAF1 polypeptidesand is described in detail in Geysen, PCT published application WO84/03564, published on Sep. 13, 1984. Briefly stated, large numbers ofdifferent small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with APAF1 polypeptides and washed. Bound APAF1polypeptides are then detected by methods well known in the art.

Purified APAF1 can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to capture antibodies toimmobilize the APAF1 polypeptide on the solid phase.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of specifically bindingthe APAF1 polypeptide compete with a test compound for binding to theAPAF1 polypeptide or fragments thereof. In this manner, the antibodiescan be used to detect the presence of any peptide which shares one ormore antigenic determinants of the APAF1 polypeptide.

A further technique for drug screening involves the use of hosteukaryotic cell lines or cells (such as described above) which express awild-type or mutant APAF1 gene and as a consequence of expression ofwild type or mutant APAF1 demonstrate a specific phenotype. Thephenotype of the cells is examined to determine if the compound iscapable of modulating the phenotype and thereby APAF1 function.

Briefly, a method of screening for a substance which modulates activityof a polypeptide may include contacting one or more test substances withthe polypeptide in a suitable reaction medium, testing the activity ofthe treated polypeptide and comparing that activity with the activity ofthe polypeptide in comparable reaction medium untreated with the testsubstance or substances. A difference in activity between the treatedand untreated polypeptides is indicative of a modulating effect of therelevant test substance or substances.

Prior to or as well as being screened for modulation of activity, testsubstances may be screened for ability to interact with the polypeptide,e.g., in a yeast two-hybrid system (e.g., Bartel, P. L., et al. (1993)In: Cellular Interactions in Development: A Practical Approach, OxfordUniversity Press, pp. 153 179; Fields, S, and Song, O-K. (1989) Nature340:245 246; Chevray, P. M. and Nathans, D. N. (1992) Proc. Natl. Acad.Sci. USA 89:5789 5793; Lee, J. E., et al. (1995) Science 268:836 844).This system can be used as a coarse screen prior to testing a substancefor actual ability to modulate activity of the polypeptide.Alternatively, the screen could be used to screen test substances forbinding to an APAF1 specific binding partner, or to find mimetics of anAPAF1 polypeptide.

In another embodiment, the invention provides methods of screeninginhibitors of APAF1 activity or an altered APAF1 activity. In one aspectof this embodiment, the assays are configured to identify compounds thatinhibit the ability of APAF1 to activate apoptosis. The ability of APAF1to activate apoptosis can be determined by, e.g., analyzing the abilityof APAF1 to activate caspase-9 or other caspases involved in activatingapoptosis. The APAF1 based screening can employ wild-type APAF1 proteinor, preferably mutant APAF1 proteins as disclosed herein.

The invention provides screening assays for identifying inhibitors thatdisrupt the interaction between APAF1, particularly an altered (mutant)APAF1, and its interacting partners such as APAF1, AT1, caspases (e.g.,pro-caspase-9 and caspase-9), and/or cytochrome c. According to thisembodiment, the screening methods are configured for selectingmodulators of a protein complex formed between APAF1 or a homologue,derivative, altered (mutant) form, or fragment thereof and at least oneprotein with which it interacts to (or a homologue, derivative, alteredform, or fragment thereof). In a preferred aspect of this embodiment,the screening assays are configured to identify compounds that modulatean interaction between an altered APAF1 and at least one protein withwhich it interacts to activate apoptosis. For example, in a specificaspect, a screen can involve using an APAF1-cytochrome c multimericapoptosome complex based assay to examine the affect of test compoundsto modulate procaspase-9 activation (Zou et al. (1999) J. Biol. Chem.274:11549 11556). More specifically, this screening assay involvescontacting a test compound with a solution comprising, APAF1 (or analtered APAF1), cytochrome c, ATP or dATP, and procaspase-9, all ofwhich are readily available to the skilled artisan. After incubation foran appropriate amount of time, the solution can be analyzed for theformation of caspase-9 from procaspase-9 using known techniques, oralternative the assay can also include procaspase-3, and the assay canbe analyzed for the formation of caspase-3 from procaspase-3 using knowntechniques (caspase-3 activity assays). Accordingly, the screen can usecomplexes comprising wild-type APAF1 or altered APAF1, to examine theeffect of test compounds on procaspase-9 (or procaspase-3) activation.Comparing the results between the wild-type APAF1 assays and the assayshaving an altered APAF1 can allow for the identification of drugcandidates that selectively modulate altered APAF1 and/or its activationof the caspase cascade. Screening methods are also provided forselecting modulators of APAF1. The compounds identified in the screeningmethods of the present invention can be used in preventing orameliorating depression and related disorders.

Thus, test compounds can be screened in an in vitro binding assay toidentify compounds capable of binding or affecting a protein-proteininteraction between APAF1 (including homologues, derivatives, altered(mutant) forms or fragments thereof) and proteins with which itinteracts, such as APAF1, an APAF1 receptor (AT1), procaspase-9,caspase-9, and/or cytochrome c (including homologues, derivatives,altered (mutant) forms, or fragments thereof). In addition, in vitrodissociation assays may also be employed to select compounds capable ofdissociating or destabilizing the protein complexes comprising APAF1 (ormutant APAF1) identified in accordance with the present invention. An invitro screening assay can also be used to identify compounds thattrigger or initiate the formation of, or stabilize, a protein complex ofthe present invention. In preferred embodiments, in vivo assays such asyeast two-hybrid assays and various derivatives thereof, preferablyreverse two-hybrid assays, are utilized in identifying compounds thatinterfere with or disrupt protein-protein interactions between APAF1 ora homologue, derivative, altered form, mutant, or fragment thereof andan interacting partner which it interacts with such as APAF1, an APAF1receptor (AT1), procaspase-9, caspase-9, and/or cytochrome c (or ahomologue, derivative, altered form, mutant, or fragment thereof). Inaddition, systems such as yeast two-hybrid assays are also useful inselecting compounds capable of triggering or initiating, enhancing orstabilizing protein-protein interactions between APAF1 or a homologue,derivative, altered form, mutant, or fragment thereof and a protein withwhich it interacts, such as an APAF1, APAF1 receptor (AT1),procaspase-9, caspase-9, or cytochrome c (or a homologue, derivative,altered form, mutant, or fragment thereof). For example, the assays canentail (1) contacting the interacting members of the protein complexwith each other in the presence of a test compound; and (2) detectingthe interaction between the interacting members.

The test compounds may be screened in an in vitro assay to identifycompounds capable of binding the protein complexes or interactingprotein members thereof in accordance with the present invention. Forthis purpose, a test compound is contacted with a protein complex or aninteracting protein member thereof under conditions and for a timesufficient to allow specific interaction between the test compound andthe target components to occur, thereby resulting in the binding of thecompound to the target, and the formation of a complex. Subsequently,the binding event is detected. Various screening techniques known in theart may be used in the present invention. The protein complexes and theinteracting protein members thereof may be prepared by any suitablemethods, e.g., by recombinant expression and purification. The proteincomplexes and/or interacting protein members thereof (both are referredto as “target” hereinafter in this section) may be free in solution. Atest compound may be mixed with a target forming a liquid mixture. Thecompound may be labeled with a detectable marker. Upon mixing undersuitable conditions, the binding complex having the compound and thetarget can be co-immunoprecipitated and washed. The compound in theprecipitated complex can be detected based on the marker on thecompound.

In a preferred embodiment, the target is immobilized on a solid supportor on a cell surface. Preferably, the target can be arrayed into aprotein microchip according to methods well-known in the art. Forexample, a target may be immobilized directly onto a microchip substratesuch as glass slides or onto multi-well plates using non-neutralizingantibodies, i.e., antibodies capable of binding to the target but do notsubstantially affect its biological activities. To affect the screening,test compounds can be contacted with the immobilized target to allowbinding to occur to form complexes under standard binding assayconditions. Either the targets or test compounds are labeled with adetectable marker using well-known labeling techniques. For example,U.S. Pat. No. 5,741,713 discloses combinatorial libraries of biochemicalcompounds labeled with NMR active isotopes. To identify bindingcompounds, one may measure the formation of the target-test compoundcomplexes or kinetics for the formation thereof. When combinatoriallibraries of organic non-peptide non-nucleic acid compounds arescreened, it is preferred that labeled or encoded (or “tagged”)combinatorial libraries are used to allow rapid decoding of leadstructures. This is especially important because, unlike biologicallibraries, individual compounds found in chemical libraries cannot beamplified by self-amplification. Tagged combinatorial libraries areprovided in, e.g., Borchardt and Still, J. Am. Chem. Soc., 116:373 374(1994) and Moran et al., J. Am. Chem. Soc., 117:10787 10788 (1995), bothof which are incorporated herein by reference.

Alternatively, the test compounds can be immobilized on a solid support,e.g. forming a microarray of test compounds. The target protein orprotein complex is then contacted with the test compounds. The targetcan be labeled with any suitable detection marker. For example, thetarget can be labeled with radioactive isotopes or fluorescence markerbefore binding reaction occurs. Alternatively, after the bindingreactions, antibodies that are immunoreactive with the target and arelabeled with radioactive materials, fluorescence markers, enzymes, orlabeled secondary anti-Ig antibodies can be used to detect any boundtarget thus identifying the binding compound. One example of thisembodiment is the protein probing method. That is, the target providedin accordance with the present invention is used as a probe to screenexpression libraries of proteins or random peptides. The expressionlibraries can be phage display libraries, in vitro translation-basedlibraries, or ordinary expression cDNA libraries. The libraries may beimmobilized on a solid support such as nitrocellulose filters. See e.g.,Sikela and Hahn, Proc. Natl. Acad. Sci. USA, 84:3038 3042 (1987). Theprobe can be labeled with a radioactive isotope or a fluorescencemarker. Alternatively, the probe can be biotinylated and detected with astreptavidin-alkaline phosphatase conjugate. More conveniently, thebound probe can be detected with an antibody.

In yet another embodiment, a known ligand capable of binding to thetarget can be used in competitive binding assays. Complexes between theknown ligand and the target can be formed and then contacted with testcompounds. The ability of a test compound to interfere with theinteraction between the target and the known ligand is measured. Oneexemplary ligand is an antibody capable of specifically binding thetarget. Particularly, such an antibody is especially useful foridentifying peptides that share one or more antigenic determinants ofthe target protein complex or interacting protein members thereof.

In a specific embodiment, a protein complex used in the screening assayincludes a hybrid protein which is formed by fusion of two interactingprotein members or fragments or interaction domains thereof. The hybridprotein can also be designed such that it contains a detectable epitopetag fused thereto. Suitable examples of such epitope tags includesequences derived from, e.g., influenza virus hemagglutinin (HA), SimianVirus 5 (V5), polyhistidine (6×His), c-myc, lacZ, GST, and the like.

Test compounds may also be screened in in vitro assays to identifycompounds capable of dissociating the protein complexes identified inaccordance with the present invention. Thus, for example, dissociationof a protein complex comprising APAF1 (or mutant APAF1) and at least oneinteracting partner (such as APAF1, AT1, procaspase-9, caspase-9, and/orcytochrome c) following treatment with a test compound can be detected.Conversely, test compounds may also be screened to identify compoundscapable of enhancing the interaction between APAF1 (or altered APAF1)and at least one interacting partner (such as AT1, procaspase-9,caspase-9, and/or cytochrome c) or stabilizing the protein complexformed by the proteins.

The assay can be conducted in similar manners as the binding assaysdescribed above. For example, the presence or absence of a particularprotein complex can be detected by an antibody selectivelyimmunoreactive with the protein complex. Thus, after incubation of theprotein complex with a test compound, an immunoprecipitation assay canbe conducted with the antibody. If the test compound disrupts theprotein complex, then the amount of immunoprecipitated protein complexin this assay will be significantly less than that in a control assay inwhich the same protein complex is not contacted with the test compound.Similarly, two proteins the interaction between which is to be enhancedcan be incubated together with a test compound. Thereafter, a proteincomplex can be detected by the selectively immunoreactive antibody. Theamount of protein complex can be compared to that formed in the absenceof the test compound. Various other detection methods can be suitable inthe dissociation assay, as will be apparent to a skilled artisanapprised of the present disclosure.

Protein complexes comprising APAF1 (or mutant APAF1) and an interactingpartner including APAF1, an APAF1 receptor (AT1), procaspase-9,caspase-9 and/or cytochrome c, can be used in screening assays toidentify modulators of protein complexes comprising APAF1. In addition,mutants, homologues, derivatives or fragments of APAF1 and proteincomplexes containing such homologues, derivatives, mutants, or fragmentsmay also be used in such screening assays. As used herein, the term“modulator” encompasses any compounds that can cause any form ofalteration of the biological activities or functions of the proteins orprotein complexes, including, e.g., enhancing or reducing theirbiological activities, increasing or decreasing their stability,altering their affinity or specificity to certain other biologicalmolecules, etc. In addition, the term “modulator” as used herein alsoincludes any compounds that simply bind APAF1, mutant APAF1, and/or theproteins complexes of the present invention. For example, a modulatorcan be an “interaction antagonist” capable of interfering with ordisrupting or dissociating protein-protein interaction between APAF1 ora homologue, fragment or derivative thereof. A modulator can also be an“interaction agonist” that initiates or strengthens the interactionbetween the protein members of the protein complex of the presentinvention, or homologues, fragments, mutants, or derivatives thereof.

Accordingly, the present invention provides screening methods forselecting modulators of APAF1 or an altered form thereof, or proteincomplexes formed between an APAF1 receptor (AT1), procaspase-9,caspase-9, and/or cytochrome c or a mutant form thereof. The proteincomplex targets suitable in the screening assays of the presentinvention can be any embodiments of the protein complexes of the presentinvention. Preferably, protein fragments are used in forming the proteincomplexes. In specific embodiments, fusion proteins are used in which adetectable epitope tag is fused to an interacting protein or a homologueor derivative or fragment thereof. Suitable examples of such epitopetags include sequences derived from, e.g., influenza virus hemagglutinin(HA), Simian Virus 5 (V5), polyhistidine (6×His), c-myc, lacZ, GST, andthe like. In addition, an interacting protein alone or a homologue orderivative or fragment thereof can also be used as a protein target inscreening assays. Preferably, a detectable epitope tag is fused to theprotein target. For example, compounds capable of binding to APAF1protein, a homologue, derivative, mutant, or fragment thereof selectedby the screening assays can be tested for their ability to inhibit orinterfere with the interactions between APAF1 and APAF1, an APAF1receptor (AT1), APAF1, procaspase-9, caspase-9, and/or cytochrome c.

The modulators selected in accordance with the screening methods of thepresent invention can be effective in modulating the functions oractivities of APAF1 alone, or the protein complexes comprising APAF1 ormutant APAF1. Such complexes can include an APAF1 receptor (AT1), APAF1,procaspase-9, caspase-9, and/or cytochrome c. For example, compoundscapable of binding the protein complexes can be capable of modulatingthe functions of the protein complexes. Additionally, compounds thatinterfere with, weaken, dissociate or disrupt, or alternatively,initiate, facilitate or stabilize the protein-protein interactionbetween the interacting protein members of the protein complexes canalso be effective in modulating the functions or activities of theprotein complexes. Thus, the compounds identified in the screeningmethods of the present invention can be made into therapeutically orprophylactically effective drugs for preventing or amelioratingdiseases, disorders or symptoms caused by or associated with proteincomplexes comprising APAF1 (or mutant APAF1) and APAF1 (or mutant APAF)an APAF1 receptor (AT1), procaspase-9, caspase-9, and/or cytochrome c,or APAF1 separately. Alternatively, they can be used as leads to aid thedesign and identification of therapeutically or prophylacticallyeffective compounds for diseases, disorders or symptoms caused by orassociated with protein complexes comprising APAF1 (or an mutant APAF1)and APAF1 (or an mutant APAF1), an APAF1 receptor (AT1), procaspase-9,caspase-9, and/or cytochrome c, or APAF1 separately. The proteincomplexes and/or interacting protein members thereof in accordance withthe present invention can be used in any of a variety of drug screeningtechniques. Drug screening can be performed as described herein or usingwell-known techniques, such as those described in U.S. Pat. Nos.5,800,998 and 5,891,628, both of which are incorporated herein byreference.

In addition, potentially useful agents also include incomplete proteins,i.e., fragments of the interacting protein members that are capable ofbinding to their respective binding partners in a protein complex butare defective with respect to their normal cellular functions. Forexample, binding domains of the interacting member proteins of a proteincomplex can be used as competitive inhibitors of the activities of theprotein complex. As will be apparent to skilled artisans, derivatives,homologues, or mutants of the binding domains can also be used. Bindingdomains can be easily identified using molecular biology techniques,e.g., mutagenesis in combination with yeast two-hybrid assays.Preferably, the protein fragment used is a fragment of an interactingprotein member having a length of less than 90%, 80%, more preferablyless than 75%, 65%, 50%, or less than 40% of the full length of theprotein member. In one embodiment, an APAF1 receptor (AT1),procaspase-9, caspase-9, or cytochrome c protein fragment isadministered. In a specific embodiment, one or more of the interactiondomains of an APAF1 receptor (AT1), procaspase-9, caspase-9, orcytochrome c are administered to cells or tissue in vitro, or areadministered to a patient in need of such treatment. For example,suitable protein fragments can include polypeptides having a contiguousspan of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20 or 25,preferably from 4 to 30, 40 or 50 amino acids or more of the sequence ofan APAF1 receptor (AT1), procaspase-9, caspase-9, or cytochrome c thatare capable of interacting with APAF1 or mutant APAF1. Also, suitableprotein fragments can also include peptides capable of binding APAF1, ormutant APAF1, and having an amino acid sequence of from 4 to 30 aminoacids that is at least 75%, 80%, 82%, 85%, 87%, 90%, 95% or moreidentical to a contiguous span of amino acids of an APAF1 receptor(AT1), procaspase-9, caspase-9, and/or cytochrome c of the same length.Alternatively, a polypeptide capable of interacting with an APAF1receptor (AT1), procaspase-9, caspase-9, or cytochrome c and having acontiguous span of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20 or25, preferably from 4 to 30, 40 or 50 or more amino acids of the aminoacid sequence of APAF1 can be administered. Also, other examples ofsuitable compounds include a peptide capable of binding an APAF1receptor (AT1), procaspase-9, caspase-9, or cytochrome c and having anamino acid sequence of from 4 to 30, 40, 50 or more amino acids that isat least 75%, 80%, 82%, 85%, 87%, 90%, 95% or more identical to acontiguous span of amino acids of the same length from APAF1. Inaddition, the administered compounds can also be an antibody or antibodyfragment, preferably single-chain antibody immunoreactive with APAF1 oran APAF1 receptor (AT1), procaspase-9, caspase-9, or cytochrome c or aprotein complex of the present invention.

The protein fragments suitable as competitive inhibitors can bedelivered into cells by direct cell internalization, receptor mediatedendocytosis, or via a “transporter.” It is noted that when the targetproteins or protein complexes to be modulated reside inside cells, thecompound administered to cells in vitro or in vivo in the method of thepresent invention preferably is delivered into the cells in order toachieve optimal results. Thus, preferably, the compound to be deliveredis associated with a transporter capable of increasing the uptake of thecompound by cells harboring the target protein or protein complex. Asused herein, the term “transporter” refers to an entity (e.g., acompound or a composition or a physical structure formed from multiplecopies of a compound or multiple different compounds) that is capable offacilitating the uptake of a compound of the present invention by animalcells, particularly human cells. Typically, the cell uptake of acompound of the present invention in the presence of a “transporter” isat least 20% higher, preferably at least 40%, 50%, 75%, and morepreferably at least 100% higher than the cell uptake of the compound inthe absence of the “transporter.” Many molecules and structures known inthe art can be used as “transporters.” In one embodiment, a penetratinis used as a transporter. For example, the homeodomain of Antennapedia,a Drosophila transcription factor, can be used as a transporter todeliver a compound of the present invention. Indeed, any suitable memberof the penetratin class of peptides can be used to carry a compound ofthe present invention into cells. Penetratins are disclosed in, e.g.,Derossi et al., Trends Cell Biol., 8:84 87 (1998), which is incorporatedherein by reference. Penetratins transport molecules attached theretoacross cytoplasmic membranes or nuclear membranes efficiently, in areceptor-independent, energy-independent, and cell type-independentmanner. Methods for using a penetratin as a carrier to deliveroligonucleotides and polypeptides are also disclosed in U.S. Pat. No.6,080,724; Pooga et al., Nat. Biotech., 16:857 (1998); and Schutze etal., J. Immunol., 157:650 (1996), all of which are incorporated hereinby reference. U.S. Pat. No. 6,080,724 defines the minimal requirementsfor a penetratin peptide as a peptide of 16 amino acids with 6 to 10 ofwhich being hydrophobic. The amino acid at position 6 counting fromeither the N- or C-terminus is tryptophan, while the amino acids atpositions 3 and 5 counting from either the N- or C-terminus are not bothvaline. Preferably, the helix 3 of the homeodomain of DrosophilaAntennapedia is used as a transporter. More preferably, a peptide havinga sequence of amino acid residues 43 58 of the homeodomain Antp isemployed as a transporter. In addition, other naturally occurringhomologs of the helix 3 of the homeodomain of Drosophila Antennapediacan be used. For example, homeodomains of Fushi-tarazu and Engrailedhave been shown to be capable of transporting peptides into cells. SeeHan et al., Mol. Cells, 10:728 32 (2000). As used herein, the term“penetratin” also encompasses peptoid analogs of the penetratinpeptides. Typically, the penetratin peptides and peptoid analogs thereofare covalently linked to a compound to be delivered into cells thusincreasing the cellular uptake of the compound.

In another embodiment, the HIV-1 tat protein or a derivative thereof isused as a “transporter” covalently linked to a compound according to thepresent invention. The use of HIV-1 tat protein and derivatives thereofto deliver macromolecules into cells has been known in the art. SeeGreen and Loewenstein, Cell, 55:1179 (1988); Frankel and Pabo, Cell,55:1189 (1988); Vives et al., J. Biol. Chem., 272:16010 16017 (1997);Schwarze et al., Science, 285:1569 1572 (1999). It is known that thesequence responsible for cellular uptake consists of the highly basicregion, amino acid residues 49 57. See e.g., Vives et al., J. Biol.Chem., 272:16010 16017 (1997); Wender et al., Proc. Nat'l Acad. Sci.USA, 97:13003 13008 (2000). The basic domain is believed to target thelipid bilayer component of cell membranes. It causes a covalently linkedprotein or nucleic acid to cross cell membrane rapidly in a celltype-independent manner. Proteins ranging in size from 15 to 120 kD havebeen delivered with this technology into a variety of cell types both invitro and in vivo. See Schwarze et al., Science, 285:1569 1572 (1999).Any HIV tat-derived peptides or peptoid analogs thereof capable oftransporting macromolecules such as peptides can be used for purposes ofthe present invention. For example, any native tat peptides having thehighly basic region, amino acid residues 49 57 can be used as atransporter by covalently linking it to the compound to be delivered. Inaddition, various analogs of the tat peptide of amino acid residues 4957 can also be useful transporters for purposes of this invention.Examples of various such analogs are disclosed in Wender et al., Proc.Nat'l Acad. Sci. USA, 97:13003 13008 (2000) (which is incorporatedherein by reference) including, e.g., d-Tat₄₉₋₅₇, retro-inverso isomersof l- or d-Tat₄₉₋₅₇ (i.e., l-Tat₅₇₋₄₉ and d-Tat₅₇₋₄₉), L-arginineoligomers, D-arginine oligomers, L-lysine oligomers, D-lysine oligomers,L-histidine oligomers, D-histidine oligomers, L-ornithine oligomers,D-ornithine oligomers, and various homologues, derivatives (e.g.,modified forms with conjugates linked to the small peptides) and peptoidanalogs thereof.

Other useful transporters known in the art include, but are not limitedto, short peptide sequences derived from fibroblast growth factor (SeeLin et al., J. Biol. Chem., 270:14255 14258 (1998)), Galparan (See Poogaet al., FASEB J. 12:67 77 (1998)), and HSV-1 structural protein VP22(See Elliott and O'Hare, Cell, 88:223 233 (1997)). As theabove-described various transporters are generally peptides, fusionproteins can be conveniently made by recombinant expression to contain atransporter peptide covalently linked by a peptide bond to a competitiveprotein fragment. Alternatively, conventional methods can be used tochemically synthesize a transporter peptide or a peptide of the presentinvention or both.

The hybrid peptide can be administered to cells or tissue in vitro or topatients in a suitable pharmaceutical composition. In addition topeptide-based transporters, various other types of transporters can alsobe used, including but not limited to cationic liposomes (see Rui etal., J. Am. Chem. Soc., 120:11213 11218 (1998)), dendrimers (Kono etal., Bioconjugate Chem., 10:1115 1121 (1999)), siderophores (Ghosh etal., Chem. Biol., 3:1011 1019 (1996)), etc. In a specific embodiment,the compound according to the present invention is encapsulated intoliposomes for delivery into cells.

Additionally, when a compound according to the present invention is apeptide, it can be administered to cells by a gene therapy method. Thatis, a nucleic acid encoding the peptide can be administered to cells invitro or to cells in a human or animal body. Any suitable gene therapymethods may be used for purposes of the present invention. Various genetherapy methods are well known in the art. Successes in gene therapyhave been reported recently. See e.g., Kay et al., Nature Genet., 24:25761 (2000); Cavazzana-Calvo et al., Science, 288:669 (2000); and Blaeseet al., Science, 270: 475 (1995); Kantoff, et al., J. Exp. Med., 166:219(1987).

Methods of Use: Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact (e.g., agonists, antagonists, inhibitors) in orderto fashion drugs which are, for example, more active or stable forms ofthe polypeptide, or which, e.g., enhance or interfere with the functionof a polypeptide in vivo. See, e.g., Hodgson, J. (1991) Bio/Technology9:19 21. Rational drug design can be performed as described herein orusing well known techniques, such as described in U.S. Pat. Nos.5,800,998 and 5,891,628, each incorporated herein by reference.

In one approach, one first determines the three-dimensional structure ofa protein of interest (e.g., APAF1 polypeptide, fragments of the APAF1polypeptide, or apoptosome (Acehan et al. Mol. Cell. 9:423 432 (2002)))or, for example, of the APAF1-receptor or ligand complex, by x-raycrystallography, by computer modeling or most typically, by acombination of approaches. Less often, useful information regarding thestructure of a polypeptide may be gained by modeling based on thestructure of homologous proteins. An example of rational drug design isthe development of HIV protease inhibitors (Erickson, J. et al., (1990)Science 249:527 533). In addition, peptides (e.g., APAF1 polypeptide orfragments thereof) are analyzed by an alanine scan (Wells, J. A. (1991)Methods in Enzymol. 202:390 411). In this technique, an amino acidresidue is replaced by Ala, and its effect on the peptide's activity isdetermined. Each of the amino acid residues of the peptide is analyzedin this manner to determine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by afunctional assay, and then to solve its crystal structure. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies (anti-ids) to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of the anti-ids would be expected to be an analog ofthe original receptor. The anti-id could then be used to identify andisolate peptides from banks of chemically or biologically produced banksof peptides. Selected peptides would then act as the pharmacore. Thus,one may design drugs which have, e.g., improved APAF1 polypeptideactivity or stability or which act as inhibitors, agonists, antagonists,etc. of APAF1 polypeptide activity.

Following identification of a substance which modulates or affectspolypeptide activity, the substance can be investigated further.Furthermore, it can be manufactured and/or used in preparation, i.e.,manufacture or formulation, or a composition such as a medicament,pharmaceutical composition or drug. These substances can be administeredto individuals.

Thus, the present invention extends in various aspects not only to asubstance identified using a nucleic acid molecule as a modulator ofpolypeptide activity, in accordance with what is disclosed herein, butalso a pharmaceutical composition, medicament, drug or other compositioncomprising such a substance, a method comprising administration of sucha composition comprising such a substance, a method comprisingadministration of such a composition to a patient, e.g., for treatmentor prophylaxis of depression, use of such a substance in the manufactureof a composition for administration, e.g., for treatment or prophylaxisof depression, and a method of making a pharmaceutical compositioncomprising admixing such a substance with a pharmaceutically acceptableexcipient, vehicle or carrier, and optionally other ingredients.

A substance identified as a modulator of polypeptide function can bepeptide or non-peptide in nature. Non-peptide “small molecules” areoften preferred for many in vivo pharmaceutical uses. Accordingly, amimetic or mimic of the substance (particularly if a peptide) may bedesigned for pharmaceutical use.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesize or where it is unsuitable for a particularmethod of administration, e.g., pure peptides are unsuitable activeagents for oral compositions as they tend to be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis and testingis generally used to avoid randomly screening large numbers of moleculesfor a target property.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. First, the particular parts ofthe compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g., by substituting each residue in turn. Alanine scans of peptide arecommonly used to refine such peptide motifs. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modeledaccording to its physical properties, e.g., stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.,spectroscopic techniques, x-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modeling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modeled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted onto it can conveniently be selected so that themimetic is easy to synthesize, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. Alternatively, where the mimetic ispeptide-based, further stability can be achieved by cyclizing thepeptide, increasing its rigidity. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimization ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

Methods of Use: Nucleic Acid Based Therapies

According to the present invention, a method is also provided ofsupplying wild-type APAF1 function to a cell which carries mutant APAF1alleles. The wild-type APAF1 gene or a part of the gene can beintroduced into the cell in a vector such that the gene remainsextrachromosomal. In such a situation, the gene will be expressed by thecell from the extrachromosomal location. If a gene fragment isintroduced and expressed in a cell carrying a mutant APAF1 allele, thegene fragment should encode a part of the APAF1 protein which isrequired for normal physiological processes of the cell. More preferredis the situation where the wild-type APAF1 gene or a part thereof isintroduced into the mutant cell in such a way that it recombines withthe endogenous mutant APAF1 gene present in the cell. Such recombinationrequires a double recombination event which results in the correction ofthe APAF1 gene mutation. Vectors for introduction of genes both forrecombination and for extrachromosomal maintenance are known in the art,and any suitable vector may be used. Methods for introducing DNA intocells such as electroporation, calcium phosphate coprecipitation andviral transduction are known in the art, and the choice of method iswithin the competence of the routineer. See also U.S. Pat. Nos.5,800,998 and 5,891,628, each incorporated by reference herein.

Among the compounds which may exhibit anti-depression activity areantisense, siRNA, ribozyme, and triple helix molecules. Such moleculesmay be designed to reduce or inhibit mutant APAF1 activity. Techniquesfor the production and use of such molecules are well known to those ofskill in the art, such as described herein or in U.S. Pat. No.5,800,998, incorporated herein by reference.

Antisense RNA and DNA molecules act to directly block the translation ofmRNA by binding to targeted mRNA and preventing protein translation.With respect to antisense DNA, oligodeoxyribonucleotides derived fromthe translation initiation site, e.g., between the −10 and +10 regionsof the APAF1 nucleotide sequence of interest, are preferred.

In one embodiment, the inhibitors of cellular levels of APAF1 aredouble-stranded small interfering RNA (siRNA) compounds or a modifiedequivalent thereof. APAF1 siRNA are commercially available (targetsequence=SEQ ID NO:45′-AAT TGG TGC ACT TTT ACG TGA-3′) from, forexample, Dharmacon (Lafayette, Colo.) (citing Lassus et al. Science297:1352 1354 (2002)). Alternatively, the skilled artisan, apprised ofthis disclosure, is capable of providing siRNA useful for reducing thelevels of APAF1 protein (or mutants thereof).

As is generally known in the art, siRNA compounds are RNA duplexescomprising two complementary single-stranded RNAs of 21 nucleotides thatform 19 base pairs and possess 3′ overhangs of two nucleotides. SeeElbashir et al., Nature 411:494 498 (2001); and PCT Publication Nos. WO00/44895; WO 01/36646; WO 99/32619; WO 00/01846; WO 01/29058; WO99/07409; and WO 00/44914. When appropriately targeted via itsnucleotide sequence to a specific mRNA in cells, a siRNA canspecifically suppress gene expression through a process known as RNAinterference (RNAi). See e.g., Zamore & Aronin, Nature Medicine, 9:266267 (2003). siRNAs can reduce the cellular level of specific mRNAs, anddecrease the level of proteins coded by such mRNAs. siRNAs utilizesequence complementarity to target an mRNA for destruction, and aresequence-specific. Thus, they can be highly target-specific, and inmammals have been shown to target mRNAs encoded by different alleles ofthe same gene. Because of this precision, side effects typicallyassociated with traditional drugs can be reduced or eliminated. Inaddition, they are relatively stable, and like antisense and ribozymemolecules, they can also be modified to achieve improved pharmaceuticalcharacteristics, such as increased stability, deliverability, and easeof manufacture. Moreover, because siRNA molecules take advantage of anatural cellular pathway, i.e., RNA interference, they are highlyefficient in destroying targeted mRNA molecules. As a result, it isrelatively easy to achieve a therapeutically effective concentration ofan siRNA compound in patients. Thus, siRNAs are a new class of drugsbeing actively developed by pharmaceutical companies.

In vivo inhibition of specific gene expression by RNAi was achieved invarious organisms including mammals. For example, Song et al., NatureMedicine, 9:347 351 (2003) demonstrate that intravenous injection of FassiRNA compounds into laboratory mice with autoimmune hepatitisspecifically reduced Fas mRNA levels and expression of Fas protein inmouse liver cells. The gene silencing effect persisted withoutdiminution for 10 days after the intravenous injection. The injectedsiRNA was effective in protecting the mice from liver failure andfibrosis. Song et al., Nature Medicine, 9:347 351 (2003). Several otherapproaches for delivery of siRNA into animals have also proved to besuccessful. See e.g., McCaffery et al., Nature, 418:38 39 (2002); Lewiset al., Nature Genetics, 32:107 108 (2002); and Xia et al., NatureBiotech., 20:1006 1010 (2002).

The siRNA compounds provided according to the present invention can besynthesized using conventional RNA synthesis methods. For example, theycan be chemically synthesized using appropriately protectedribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.Various applicable methods for RNA synthesis are disclosed in, e.g.,Usman et al., J. Am. Chem. Soc., 109:7845 7854 (1987) and Scaringe etal., Nucleic Acids Res., 18:5433 5441 (1990). Custom siRNA synthesisservices are available from commercial vendors such as Ambion (Austin,Tex., USA), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical(Rockford, Ill., USA), ChemGenes (Ashland, Mass., USA), Proligo(Hamburg, Germany), and Cruachem (Glasgow, UK).

The siRNA compounds can also be various modified equivalents of thestructures in of any APAF1 siRNA. As used herein, “modified equivalent”means a modified form of a particular siRNA compound having the sametarget-specificity (i.e., recognizing the same mRNA molecules thatcomplement the unmodified particular siRNA compound). Thus, a modifiedequivalent of an unmodified siRNA compound can have modifiedribonucleotides, that is, ribonucleotides that contain a modification inthe chemical structure of an unmodified nucleotide base, sugar and/orphosphate (or phosphodiester linkage). As is known in the art, an“unmodified ribonucleotide” has one of the bases adenine, cytosine,guanine, and uracil joined to the 1′ carbon of beta-D-ribo-furanose.

Preferably, modified siRNA compounds contain modified backbones ornon-natural internucleoside linkages, e.g., modifiedphosphorous-containing backbones and non-phosphorous backbones such asmorpholino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate,sulfonamide, and sulfamate backbones; formacetyl and thioformacetylbackbones; alkene-containing backbones; methyleneimino andmethylenehydrazino backbones; amide backbones, and the like.

Examples of modified phosphorous-containing backbones include, but arenot limited to phosphorothioates, phosphorodithioates, chiralphosphorothioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, thionoalkylphosphonates, phosphinates, phosphoramidates,thionophosphoramidates, thionoalkylphosphotriesters, andboranophosphates and various salt forms thereof. See e.g., U.S. Pat.Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and5,625,050, each of which is herein incorporated by reference.

Examples of the non-phosphorous containing backbones described above aredisclosed in, e.g., U.S. Pat. Nos. 5,034,506; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,677,437; and 5,677,439, each of which is herein incorporated byreference.

Modified forms of siRNA compounds can also contain modified nucleosides(nucleoside analogs), i.e., modified purine or pyrimidine bases, e.g.,5-substituted pyrimidines, 6-azapyrimidines, pyridin-4-one,pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine(e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), 2-thiouridine, 4-thiouridine,5(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,5-methylaminomethyluridine, 5-methylcarbonylmethyluridine,5-methyloxyuridine, 5-methyl-2-thiouridine, 4-acetylcytidine,3-methylcytidine, propyne, quesosine, wybutosine, wybutoxosine,beta-D-galactosylqueosine, N-2, N-6 and O-substituted purines, inosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,2-methyladenosine, 2-methylguanosine, N6-methyladenosine,7-methylguanosine, 2-methylthio-N-6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives, and the like. See e.g., U.S. Pat. Nos. 3,687,808;4,845,205; 5,130,302; 5,175,273; 5,367,066; 5,432,272; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,587,469; 5,594,121; 5,596,091;5,681,941; and 5,750,692, PCT Publication No. WO 92/07065; PCTPublication No. WO 93/15187; and Limbach et al., Nucleic Acids Res.,22:2183 (1994), each of which is incorporated herein by reference in itsentirety.

In addition, modified siRNA compounds can also have substituted ormodified sugar moieties, e.g., 2′-O-methoxyethyl sugar moieties. Seee.g., U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,567,811; 5,576,427; 5,591,722;5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; and5,700,920, each of which is herein incorporated by reference.

Modified siRNA compounds may be synthesized by the methods disclosed in,e.g., U.S. Pat. No. 5,652,094; International Publication Nos. WO91/03162; WO 92/07065 and WO 93/15187; European Patent Application No.92110298.4; Perrault et al., Nature, 344:565 (1990); Pieken et al.,Science, 253:314 (1991); and Usman and Cedergren, Trends in Biochem.Sci., 17:334 (1992).

Preferably, the 3′ overhangs of the siRNAs of the present invention aremodified to provide resistance to cellular nucleases. In one embodimentthe 3′ overhangs comprise 2′-deoxyribonucleotides. In a preferredembodiment (depicted in FIGS. 4 26) these 3′ overhangs comprise adinucleotide made of two 2′-deoxythymine residues (i.e., dTdT) linked bya 5′-3′ phosphodiester linkage.

siRNA compounds may be administered to mammals by various methodsthrough different routes. For example, they can be administered byintravenous injection. See Song et al., Nature Medicine, 9:347 351(2003). They can also be delivered directly to a particular organ ortissue by any suitable localized administration methods. Several otherapproaches for delivery of siRNA into animals have also proved to besuccessful. See e.g., McCaffery et al., Nature, 418:38 39 (2002); Lewiset al., Nature Genetics, 32:107 108 (2002); and Xia et al., NatureBiotech., 20:1006 1010 (2002). Alternatively, they may be deliveredencapsulated in liposomes, by iontophoresis, or by incorporation intoother vehicles such as hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesive microspheres.

In addition, they can also be delivered by a gene therapy approach,using a DNA vector from which siRNA compounds in, e.g., small hairpinform (shRNA), can be transcribed directly. Recent studies havedemonstrated that while double-stranded siRNAs are very effective atmediating RNAi, short, single-stranded, hairpin-shaped RNAs can alsomediate RNAi, presumably because they fold into intramolecular duplexesthat are processed into double-stranded siRNAs by cellular enzymes. Suiet al., Proc. Natl. Acad. Sci. U.S.A., 99:5515 5520 (2002); Yu et al.,Proc. Natl. Acad. Sci. U.S.A., 99:6047 6052 (2002); and Paul et al.,Nature Biotech., 20:505 508 (2002)). This discovery has significant andfar-reaching implications, since the production of such shRNAs can bereadily achieved in vivo by transfecting cells or tissues with DNAvectors bearing short inverted repeats separated by a small number of(e.g., 3 to 9) nucleotides that direct the transcription of such smallhairpin RNAs. Additionally, if mechanisms are included to direct theintegration of the transcription cassette into the host cell genome, orto ensure the stability of the transcription vector, the RNAi caused bythe encoded shRNAs, can be made stable and heritable. Not only have suchtechniques been used to “knock down” the expression of specific genes inmammalian cells, but they have now been successfully employed to knockdown the expression of exogenously expressed transgenes, as well asendogenous genes in the brain and liver of living mice. See generallyHannon, Nature. 418:244 251 (2002) and Shi, Trends Genet., 19:9 12(2003); see also Xia et al., Nature Biotech., 20:1006 1010 (2002).

Additional siRNA compounds targeted at different sites of the mRNAcorresponding to APAF 1 can also be designed and synthesized accordingto general guidelines provided herein and generally known to skilledartisans. See e.g., Elbashir, et al. (Nature 411: 494 498 (2001). Forexample, guidelines have been compiled into “The siRNA User Guide” whichis available at the following web address:www.mpibpc.gwdg.de/abteilungen/100/105/sima.html.

Additionally, to assist in the design of siRNAs for the efficientRNAi-mediated silencing of any target gene, several siRNA supplycompanies maintain web-based design tools that utilize these generalguidelines for “picking” siRNAs when presented with the mRNA or codingDNA sequence of the target gene. Examples of such tools can be found atthe web sites of Dharmacon, Inc. (Lafayette, Colo.), Ambion, Inc.(Austin, Tex.), an of approximately 30 50%; (3) lack of trinucleotiderepeats, especially GGG and CCC, and (4) being unique to the target gene(i.e., sequences that share no significant homology with genes otherthan the one being targeted), so that other genes are not inadvertentlytargeted by the same siRNA designed for this particular target sequence.Another criterion to be considered is whether or not the target sequenceincludes a known polymorphic site. If so, siRNAs designed to target oneparticular allele may not effectively target another allele, sincesingle base mismatches between the target sequence and its complementarystrand in a given siRNA can greatly reduce the effectiveness of RNAiinduced by that siRNA. Given that target sequence and such design toolsand design criteria, an ordinarily skilled artisan apprised of thepresent disclosure should be able to design and synthesized additionalsiRNA compounds useful in reducing the mRNA level and therefore APAF1protein level which can be used to treat depression according to theinvention.

In another embodiment, the inhibitors of cellular levels of APAF1 areantisense compounds, or a modified equivalent thereof. U.S. Pat. No.6,468,795, which is hereby incorporated by reference in its entirety,discloses APAF1 antisense compounds and methods of modulating APAF1.These antisense compounds and methods can be employed to treat and/orprevent depression according to the therapeutic methods of theinvention. The antisense compounds according to this embodimentspecifically inhibit the expression of APAF1. As is known in the art,antisense drugs generally act by hybridizing to a particular targetnucleic acid thus blocking gene expression (particularly proteintranslation from mRNA). Methods for designing antisense compounds andusing such compounds in treating diseases are well known and welldeveloped in the art. For example, the antisense drug Vitravene®(fomivirsen), a 21-base long oligonucleotide, has been successfullydeveloped and marketed by Isis Pharmaceuticals, Inc. for treatingcytomegalovirus (CMV)-induced retinitis.

In addition to the antisense compounds provided in U.S. Pat. No.6,468,795, other antisense compounds useful in inhibiting proteintranslation from the APAF1 mRNA can also be designed and prepared. Anymethods for designing and making antisense compounds may be used forpurpose of the present invention. See generally, Sanghvi et al., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993.Typically, antisense compounds are oligonucleotides designed based onthe nucleotide sequence of the host cell's protein(s) involved in viralbudding (or egress) mRNA or gene. In particular, antisense compounds canbe designed to specifically hybridize to a particular region of the hostcell's protein(s) involved in viral budding (or egress) genomic sequenceor mRNA to interfere with replication, transcription, or translation. Asused herein, the term “specifically hybridize” or variations thereofmeans a sufficient degree of complementarity or pairing between anantisense oligo and a target DNA or mRNA such that stable and specificbinding occurs therebetween. In particular, 100% complementarity orpairing is desirable but not required. Specific hybridization occurswhen sufficient hybridization occurs between the antisense compound andits intended target nucleic acids in the substantial absence ofnon-specific binding of the antisense compound to non-target sequencesunder predetermined conditions, e.g., for purposes of in vivo treatment,preferably under physiological conditions. Preferably, specifichybridization results in the interference with normal expression of thegene product encoded by the target DNA or mRNA.

For example, an antisense compound can be designed to specificallyhybridize to the replication or transcription regulatory regions of atarget gene, or the translation regulatory regions such as translationinitiation region and exon/intron junctions, or the coding regions of atarget mRNA.

As is generally known in the art, commonly used oligonucleotides areoligomers or polymers of ribonucleic acid or deoxyribonucleic acidhaving a combination of naturally-occurring purine and pyrimidine bases,sugars and covalent linkages between nucleosides including a phosphategroup in a phosphodiester linkage. However, it is noted that the term“oligonucleotides” also encompasses various non-naturally occurringmimetics and derivatives, i.e., modified forms, of naturally-occurringoligonucleotides as described below. Typically an antisense compound ofthe present invention is an oligonucleotide having from about 6 to about200, preferably from about 8 to about 30 nucleoside bases.

The antisense compounds preferably contain modified backbones ornon-natural internucleoside linkages, including, but not limited to,modified phosphorous-containing backbones and non-phosphorous backbonessuch as morpholino backbones; siloxane, sulfide, sulfoxide, sulfone,sulfonate, sulfonamide, and sulfamate backbones; formacetyl andthioformacetyl backbones; alkene-containing backbones; methyleneiminoand methylenehydrazino backbones; amide backbones, and the like.

Examples of modified phosphorous-containing backbones include, but arenot limited to phosphorothioates, phosphorodithioates, chiralphosphorothioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, thionoalkylphosphonates, phosphinates, phosphoramidates,thionophosphoramidates, thionoalkylphosphotriesters, andboranophosphates and various salt forms thereof. See e.g., U.S. Pat.Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and5,625,050, each of which is herein incorporated by reference.

Examples of the non-phosphorous containing backbones described above aredisclosed in, e.g., U.S. Pat. Nos. 5,034,506; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,677,437; and 5,677,439, each of which is herein incorporated byreference.

Another useful modified oligonucleotide is peptide nucleic acid (PNA),in which the sugar-phosphate backbone of an oligonucleotide is replacedwith an amide containing backbone, e.g., an aminoethylglycine backbone.See U.S. Pat. Nos. 5,539,082 and 5,714,331; and Nielsen et al., Science,254, 1497 1500 (1991), all of which are incorporated herein byreference. PNA antisense compounds are resistant to RNAse H digestionand thus exhibit longer half-lives within cells. In addition, variousmodifications may be made in PNA backbones to impart desirable drugprofiles such as better stability, increased drug uptake, higheraffinity to target nucleic acid, etc.

Alternatively, the antisense compounds are oligonucleotides containingmodified nucleosides, i.e., modified purine or pyrimidine bases, e.g.,5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 andO-substituted purines, and the like. See e.g., U.S. patent Nos. as wellas U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,175,273; 5,367,066;5,432,272; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,587,469;5,594,121; 5,596,091; 5,681,941; and 5,750,692, each of which is hereinincorporated by reference in its entirety.

In addition, oligonucleotides with substituted or modified sugarmoieties may also be used. For example, an antisense compound may haveone or more 2′-O-methoxyethyl sugar moieties. See e.g., U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,567,811; 5,576,427; 5,591,722; 5,610,300; 5,627,05315,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of whichis herein incorporated by reference.

Other types of oligonucleotide modifications are also useful includinglinking an oligonucleotide to a lipid, phospholipid or cholesterolmoiety, cholic acid, thioether, aliphatic chain, polyamine, polyethyleneglycol (PEG), or a protein or peptide. The modified oligonucleotides mayexhibit increased uptake into cells, and/or improved stability, i.e.,resistance to nuclease digestion and other biodegradation. See e.g.,U.S. Pat. No. 4,522,811; Burnham, Am. J. Hosp. Pharm., 15:210 218(1994).

Antisense compounds can be synthesized using any suitable methods knownin the art. In fact, antisense compounds may be custom made bycommercial suppliers. Alternatively, antisense compounds may be preparedusing DNA/RNA synthesizers commercially available from various vendors,e.g., Applied Biosystems Group of Norwalk, Conn.

The antisense compounds can be formulated into a pharmaceuticalcomposition with suitable carriers and administered into a patient usingany suitable route of administration. Alternatively, the antisensecompounds may also be used in a “gene-therapy” approach. That is, theoligonucleotide is subcloned into a suitable vector and transformed intohuman cells. The antisense oligonucleotide is then produced in vivothrough transcription.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by an endonucleolytic cleavage. The composition ofribozyme molecules must include one or more sequences complementary tothe target APAF1 mRNA, preferably the mutant APAF1 mRNA, and mustinclude the well known catalytic sequence responsible for mRNA cleavage.For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated byreference herein in its entirety. As such, within the scope of theinvention are engineered hammerhead motif ribozyme molecules thatspecifically and efficiently catalyze endonucleolytic cleavage of RNAsequences encoding APAF1, preferably mutant APAF1 proteins.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequence: GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and ribonucleotidescorresponding to the region of the target gene containing the cleavagesite may be evaluated for predicted structural features, such assecondary structure, that may render the oligonucleotide sequenceunsuitable. The suitability of candidate targets may also be evaluatedby testing their accessibility to hybridization with complementaryoligonucleotides, using ribonuclease protection assays.

Nucleic acid molecules to be used in triplex helix formation should besingle stranded and composed of deoxynucleotides. The base compositionof these oligonucleotides must be designed to promote triple helixformation via Hoogsteen base pairing rules, which generally requiresizeable stretches of either purines or pyrimidines to be present on onestrand of a duplex. Nucleotide sequences may be pyrimidine-based, whichwill result in TAT and CGC⁺ triplets across the three associated strandsof the resulting triple helix. The pyrimidine-rich molecules providebase complementarity to a purine-rich region of a single strand of theduplex in a parallel orientation to that strand. In addition, nucleicacid molecules may be chosen that are purine-rich, for example,containing a stretch of guanidine residues. These molecules will form atriple helix with a DNA duplex that is rich in GC pairs, in which themajority of the purine residues are located on a single strand of thetargeted duplex, resulting in GGC triplets across the three strands inthe triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′,3′-5′ manner, such that they base pair with one strandof a duplex first and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

It is possible that the siRNA, antisense, ribozyme, and/or triple helixmolecules described herein may reduce or inhibit the translation of mRNAproduced by both normal and mutant APAF1 alleles. In order to ensurethat substantial normal levels of APAF1 activity are maintained in thecell, nucleic acid molecules that encode and express APAF1 polypeptidesexhibiting normal APAF1 activity can be introduced into cells which donot contain sequences susceptible to the siRNA, antisense, ribozyme, ortriple helix treatments. Such sequences can be introduced via genetherapy methods. Alternatively, it may be preferable to co-administernormal APAF1 protein into the cell or tissue in order to maintain therequisite level of cellular or tissue APAF1 activity.

Antisense RNA and DNA molecules, siRNA molecules, ribozyme molecules,and triple helix molecules of the invention can be prepared by anymethod known in the art for the synthesis of DNA and RNA molecules.These include techniques for chemically synthesizingoligodeoxyribonucleotides and oligoribonucleotides well known in the artsuch as for example solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules can be generated by in vitro and in vivotranscription of DNA sequences encoding the antisense RNA molecule. SuchDNA sequences may be incorporated into a wide variety of vectors whichincorporate suitable RNA polymerase promoters such as the T7 or SP6polymerase promoters. Alternatively, antisense cDNA constructs thatsynthesize antisense RNA constitutively or inducibly, depending on thepromoter used, can be introduced stably into cell lines.

Various well-known modifications to the DNA molecules can be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include, but are not limited to, the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

Gene therapy would be carried out according to generally acceptedmethods, for example, as described in further detail in U.S. Pat. Nos.5,837,492 and 5,800,998 and references cited therein, all incorporatedby reference herein. Expression vectors in the context of gene therapyare meant to include those constructs containing sequences sufficient toexpress a polynucleotide that has been cloned therein. In viralexpression vectors, the construct contains viral sequences sufficient tosupport packaging of the construct. If the polynucleotide encodes anantisense polynucleotide or a ribozyme, expression will produce theantisense polynucleotide or ribozyme. Thus in this context, expressiondoes not require that a protein product be synthesized. In addition tothe polynucleotide cloned into the expression vector, the vector alsocontains a promoter functional in eukaryotic cells. The clonedpolynucleotide sequence is under control of this promoter. Suitableeukaryotic promoters include those described above. The expressionvector may also include sequences, such as selectable markers and othersequences conventionally used.

Methods of Use: Peptide Therapy

Peptides which have APAF1 activity can be supplied to cells which carrymutant or missing APAF1 alleles. Peptide therapy is performed asdescribed herein or using well known techniques, such as described inU.S. Pat. Nos. 5,800,998 and 5,891,628, each incorporated herein byreference.

Protein can be produced by expression of the cDNA sequence in bacteria,for example, using known expression vectors. Alternatively, APAF1polypeptide can be extracted from APAF1-producing mammalian cells. Inaddition, the techniques of synthetic chemistry can be employed tosynthesize APAF1 protein. Any of such techniques can provide thepreparation of the present invention which comprises the APAF1 protein.Preparation is substantially free of other human proteins. This is mostreadily accomplished by synthesis in a microorganism or in vitro.

Active APAF1 molecules can be introduced into cells by microinjection orby use of liposomes, for example. Alternatively, some active moleculescan be taken up by cells, actively or by diffusion. Extracellularapplication of the APAF1 gene product can be sufficient to affect thedevelopment and or progression of depression. Supply of molecules withAPAF1 activity should lead to partial reversal of the depressionphenotype. Other molecules with APAF1 activity (for example, peptides,drugs or organic compounds) can also be used to effect such a reversal.Modified polypeptides having substantially similar function are alsoused for peptide therapy.

Alternatively, antibodies that are both specific for mutant APAF1 geneproduct and interfere with its activity can be used. Such antibodies canbe generated using standard techniques described herein or usingconventional techniques, such as described in U.S. Pat. Nos. 5,837,492;5,800,998 and 5,891,628, against the proteins themselves or againstpeptides corresponding to the binding domains of the proteins. Suchantibodies include, but are not limited to, polyclonal, monoclonal, Fabfragments, F(ab′)₂ fragments, single chain antibodies, chimericantibodies, humanized antibodies etc.

Methods of Use: Transformed Hosts; Transgenic/Knockout Animals andModels

Similarly, cells and animals which carry a mutant APAF1 allele can beused as model systems to study and test for substances which havepotential as therapeutic agents. These can be isolated from individualswith APAF1 mutations, either somatic or germline. Alternatively, thecell line can be engineered to carry the mutation in the APAF1 allele,as described above. After a test substance is applied to the cells, thephenotype of the cell is determined. Any trait of the transformed cellscan be assessed using techniques well known in the art. Transformedhosts, transgenic/knockout animals and models are prepared and used asdescribed herein or using well known techniques, such as described inU.S. Pat. Nos. 5,800,998 and 5,891,628, each incorporated herein byreference.

Animals for testing therapeutic agents can be selected after mutagenesisof whole animals or after treatment of germline cells or zygotes. Suchtreatments include insertion of mutant APAF1 alleles, usually from asecond animal species, as well as insertion of disrupted homologousgenes. Alternatively, the endogenous APAF1 gene(s) of the animals can bedisrupted by insertion or deletion mutation or other genetic alterationsusing conventional techniques (Capecchi, M. R. (1989) Science 244:1288;Valancius and Smithies, 1991; Hasty, P., K., et al. (1991) Nature350:243; Shinkai, Y., et al. (1992) Cell 68:855; Mombaerts, P., et al.(1992) Cell 68:869; Philpott, K. L., et al. (1992) Science 256:1448;Snouwaert, J. N., et al. (1992) Science 257:1083; Donehower, L. A., etal. (1992) Nature 356:215) to produce knockout or transplacementanimals. A transplacement is similar to a knockout because theendogenous gene is replaced, but in the case of a transplacement thereplacement is by another version of the same gene. After testsubstances have been administered to the animals, the depressionphenotype must be assessed. If the test substance prevents or suppressesthe depression phenotype, then the test substance is a candidatetherapeutic agent for the treatment of depression. These animal modelsprovide an extremely important testing vehicle for potential therapeuticproducts.

In one embodiment of the invention, transgenic animals are producedwhich contain a functional transgene encoding a functional APAF1polypeptide or variants thereof. Transgenic animals expressing APAF1transgenes, recombinant cell lines derived from such animals andtransgenic embryos may be useful in methods for screening for andidentifying agents that induce or repress function of APAF1. Transgenicanimals of the present invention also can be used as models for studyingindications such as depression.

In one embodiment of the invention, an APAF1 transgene is introducedinto a non-human host to produce a transgenic animal expressing a human,murine or other species APAF1 gene. The transgenic animal is produced bythe integration of the transgene into the genome in a manner thatpermits the expression of the transgene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; which is incorporated herein by reference), Brinsteret al. (1985) Mol. Cell. Biol. 8:1977 83; which is incorporated hereinby reference in its entirety) and in “Manipulating the Mouse Embryo; ALaboratory Manual” 2nd edition (eds., Hogan, Beddington, Costantimi andLong, Cold Spring Harbor Laboratory Press, 1994; which is incorporatedherein by reference in its entirety).

It can be desirable to replace the endogenous APAF1 by homologousrecombination between the transgene or a mutant gene and the endogenousgene; or the endogenous gene may be eliminated by deletion as in thepreparation of “knock-out” animals. Typically, an APAF1 gene flanked bygenomic sequences is transferred by microinjection into a fertilizedegg. The microinjected eggs are implanted into a host female, and theprogeny are screened for the expression of the transgene. Transgenicanimals can be produced from the fertilized eggs from a number ofanimals including, but not limited to reptiles, amphibians, birds,mammals, and fish. Within a particularly preferred embodiment,transgenic mice are generated which overexpress APAF1 or express amutant form of the polypeptide. Alternatively, the absence of an APAF1in “knock-out” mice permits the study of the effects that loss of APAF1protein has on a cell in vivo. Knock-out mice also provide a model forthe development of APAF 1-related depression.

Methods for producing knockout animals are generally described byShastry (Shastry et al. (1995) Experientia 51:1028 1039; Shastry et al.(1998) Mol. Cell. Biochem. 181:163 179) and Osterrieder and Wolf (1998)Rev. Sci. Tech. 17:351 364. The production of conditional knockoutanimals, in which the gene is active until knocked out at the desiredtime is generally described by Feil et al., (1996) Proc. Natl. Acad.Sci. USA 93:10887 10890; Gagneten et al. (1997) Nucl. Acids Res. 25:33263331; and Lobe & Nagy (1998) Bioessays 20:200 208. Each of thesereferences is incorporated herein by reference.

As noted above, transgenic animals and cell lines derived from suchanimals can find use in certain testing experiments. In this regard,transgenic animals and cell lines capable of expressing wild-type ormutant APAF1 can be exposed to test substances. These test substancescan be screened for the ability to alter expression of wild-type APAF1or alter the expression or function of mutant APAF1.

In a preferred aspect of the invention, compounds that are identified asmodulators of APAF1, apoptosome formation, and/or procaspase-9activation, i.e., drug candidates for treating depression, are tested inanimal or cell-based depression models. For example, a drug candidateidentified in the screening of the invention methods of the invention isfurther tested in any of the abovementioned knock-out animal models toevaluate its therapeutic effect. Alternatively, a drug candidateidentified in the screening of the invention methods of the invention isfurther tested in an animal depression model such as the forced swimtest, the tail suspension test, learned helplessness test, chronic mildtest stress, social stress test, early life stress test, olfactorybulbectomy test, fear conditioning test, anxiety based tests, rewardbased-tests, and cognition tests. See, e.g., Willner, Adv. Biochem.Psychopharmacol. 49:19 41 (1995); Porsolt, Rev. Neurosci. 11:53 58(2000); and Nestler et al. Neuron 34:13 25 (2002).

Pharmaceutical Compositions and Routes of Administration

The APAF1 polypeptides, antibodies, peptides and nucleic acids of thepresent invention can be formulated in pharmaceutical compositions,which are prepared according to conventional pharmaceutical compoundingtechniques. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990, Mack Publishing Co., Easton, Pa.). The pharmaceuticalcompositions of the invention comprise a depression therapeuticallyeffective amount of therapeutic compound. The methods of treatingdepression comprise administering to an individual in need of treatmenta therapeutically effective amount of a pharmaceutical ingredientaccording to the invention. The composition can contain the active agentor pharmaceutically acceptable salts of the active agent. Thesecompositions can comprise, in addition to one of the active substances,a pharmaceutically acceptable excipient, carrier, buffer, stabilizer orother materials well known in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The carrier can take a wide variety of forms depending onthe form of preparation desired for administration, e.g., intravenous,oral, intrathecal, epineural or parenteral.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions or emulsions. In preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, in which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets can be sugar-coated or enteric-coated bystandard techniques. The active agent can be encapsulated to make itstable to passage through the gastrointestinal tract while at the sametime allowing for passage across the blood brain barrier. See, e.g., WO96/11698.

For parenteral administration, the compound can be dissolved in apharmaceutical carrier and administered as either a solution or asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier can also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they can also be dissolved in cerebrospinal fluid.

The active agent is preferably administered in a therapeuticallyeffective amount. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofthe condition being treated. Prescription of treatment, e.g. decisionson dosage, timing, etc., is within the responsibility of generalpractitioners or specialists, and typically takes account of thedisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners. Examples of techniques and protocols can be found inRemington's Pharmaceutical Sciences 18th Ed. (1990, Mack Publishing Co.,Easton, Pa.).

Alternatively, targeting therapies can be used to deliver the activeagent more specifically to certain types of cell, by the use oftargeting systems such as antibodies or cell specific ligands. Targetingcan be desirable for a variety of reasons, e.g., if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they can be produced inthe target cell, e.g. in a viral vector such as described above or in acell based delivery system such as described in U.S. Pat. No. 5,550,050and published PCT application Nos. WO 92/19195, WO 94/25503, WO95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO96/40959 and WO 97/12635 (all of which are herein incorporated byreference), designed for implantation in a patient. The vector can betargeted to the specific cells to be treated, or it can containregulatory elements which are more tissue specific to the target cells.The cell based delivery system is designed to be implanted in apatient's body at the desired target site and contains a coding sequencefor the active agent. Alternatively, the agent can be administered in aprecursor form for conversion to the active form by an activating agentproduced in, or targeted to, the cells to be treated. See for example,EP 425,731A and WO 90/07936.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis, T., et al. (1982) Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.);Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, 2ndEd. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); Ausubel,F. M., et al. (1992) Current Protocols in Molecular Biology, (J. Wileyand Sons, NY); Glover, D. (1985) DNA Cloning, I and II (Oxford Press);Anand, R. (1992) Techniques for the Analysis of Complex Genomes,(Academic Press); Guthrie, G. and Fink, G. R. (1991) Guide to YeastGenetics and Molecular Biology (Academic Press); Harlow and Lane (1988)Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.; Jakoby, W. B. and Pastan, I. H. (eds.) (1979) CellCulture. Methods in Enzymology, Vol. 58 (Academic Press, Inc., HarcourtBrace Jovanovich (NY); Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J.Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Hogan et al. (eds) (1994) Manipulating the Mouse Embryo. ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. A general discussion of techniques andmaterials for human gene mapping, including mapping of human chromosome1, is provided, e.g., in White and Lalouel (1988) Ann. Rev. Genet.22:259 279.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and is not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 Association of APAF1 and Depression

To investigate whether mutations in APAF1 are responsible for thelinkage of depression to the chromosomal region 12q23-q24, we carriedthe analysis with the following parameters.

The phenotype used to assign affected individuals was males affectedwith either major depression or bipolar disorder. Females wereconsidered uninformative, however this does not imply that the genefound only causes depression in males. Only that the male model was usedto get the LOD score. The implication is that it is likely that thephenotype assignment is simpler in males. The model used in the analysiswas an affected only model with a dominant mode of inheritance. Theresource used to define this linkage was 84 Utah pedigrees with 1507individuals, 254 of them are affected males. The highest LOD in thestudy score is 6.1 at marker D12S1706. The region is 7 cM which is about6.5 million bases and contains approximately 20 genes. The linkage isobserved across multiple ethnic populations suggesting that this gene isa major regulator of depression and bipolar disorder and that it isacting in a critical biochemical pathway.

From fifteen families with good evidence for linkage to this region, twoaffected males who share the segregating haplotype were mutationscreened. Variants which change the encoded amino acid (missensechanges), were scrutinized for evidence that the variant changes thefunction of the gene and that the change results in a higher risk ofdepression. A gene carrying such variants would be a good candidate forthe depression susceptibility gene. Causal variants will segregate intoother affected individuals in the family and will be rarer in non-casesthan in cases.

Caspases are a highly conserved family of cysteine proteases that cleavetheir substrates after an aspartate residue. They play fundamental rolesin the initiation and execution of apoptosis. The APAF1 gene encodes aWD-repeat containing protein. WD-repeat-containing proteins are thosethat contain 4 or more copies of the WD-repeat (tryptophan-aspartaterepeat), a sequence motif approximately 31 amino acids long, thatencodes a structural repeat. Activation of procaspase-9 by the APAF1gene in the cytochrome c/dATP-dependent pathway requires proteolyticcleavage to generate the mature caspase molecule. It was shown that atruncated APAF1 variant lacking the WD-repeat domain makes APAF1constitutively active and capable of processing procaspase-9 independentof cytochrome c and dATP. Moreover the truncated protein was unable torelease the mature caspase-9 from the complex, raising the possibilitythat the WD-40 repeats play a role in the release of mature caspase-9.

Five different missense changes were detected in linked families in thisgene: Cys->Trp at amino acid 450 (C450W) in family 8546 (nucleotidechange c1350g), Gln->Arg at amino acid 465 (Q465R) in family 8428(nucleotide change a1394g), Glu->Lys at amino acid 777 (E777K) in family8347.2 (nucleotide change g2329a), Asn->Thr at amino acid 782 (A782T) infamily 8288 (nucleotide change a2345c) and Thr->Ala at amino acid 953(T953A) in family 8828803 (nucleotide change a2857g). Additionally,three other mutations have been identified corresponding to Ser357Leu;Asp479Glu; and Glu625Ala. The respective nucleotide changes are C070T;C1437G; and A1874C. Each one occurs on a haplotype that segregates intomore than one affected individual. Although these same missense changesare observed in control individuals, they are less frequent in controlsthan in cases. In addition to the linked families we are mutationscreening an additional 180 male affected cases. To date one frameshiftmutation 1299insT (inserts stop codon at codon 439) in family 8205 andanother missense change Leu->Pro at amino acid 415 (L415P) in family8582 (nucleotide change t1244c), have been seen in this random case set.Neither of these changes have been seen in 177 control samples. Theprotein encoded by the frameshift mutation is set forth in SEQ ID NO:3.

Example 2 Segregating APAF1 Mutants are Capable of Activating Caspase-9and Caspase-3 in an Apoptosome Reconstitution Assay

In order to test the affect of APAF1 mutants that segregate with majordepression an apoptosome assay was utilized. Two methods were utilizedto determine if the APAF1 segregating mutants were capable ofreconstituting a functional apoptosome. First, procaspase-9 conversionto caspase-9 was assessed by SDS-PAGE analysis and western blot analysisusing anti-caspase-9. Second, a caspase activation assay was used todetermine if procaspase-9 was converted to caspase-9 by assaying forcaspase activity.

The apoptosome reconstitution assay used in these studies was similarthat of Zou et al. J. Biol. Chem., (1999), 274(17):11549 11556. Theassay involved incubating purified cytochrome C, recombinant APAF1 orAPAF1 mutants, recombinant procaspase-9, and dATP, in a buffer having 20mM HEPES, 10 mM KCl, 2.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, pH7.5, at 30° C. After a set amount of time, the amount of conversion ofprocaspase-9 to caspase can be estimated using SDS-PAGE and western blotanalysis. These results are shown in FIG. 1 and FIG. 2. Additionally,caspase activation (indicative of procaspase-9 to caspase-9 by theapoptosome) was measured using the caspase substrate Ac-LEHD-pNA(Upstate Biotechnology, NY) in a buffer having 50 mM HEPES, 100 mM NaCl,0.1% CHAPS, 1 mM EDTA, 10% glycerol, 10 mM DTT, 100 mL total volume, 15mL apoptosome reconstitution reaction. The reaction progress wasmonitored at .DELTA.A405 nm. The results of this assay are summarized inFIG. 3.

In summary, the results of the experiments conducted according to thisexample demonstrate that the APAF1 mutants (particularly C450W, E777K,N782T, and Q465R) discovered as a result of this invention are capableenhancing activation of caspase-9 in a apoptosome activation assay ascompared to wild-type APAF1.

Example 3 Primary Apoptosome Screen for Depression Therapeutics

This example relates to identifying compounds that have potential totherapeutically effect depression. The screen of this example canutilize an apoptosome assay. Several configurations of the apoptosomeassay can be used to identify depression drug candidates. In oneconfiguration an apoptosome reconstitution assay can be used to identifycompounds that inhibit caspase activation. Wild-type APAF1 can be usedin the apoptosome assay and compounds that reduce caspase activation ascompared to control is identified as a potential depression therapeutic.Alternatively, mutant APAF1 (e.g., those discovered as a result of theinvention) can be used in the apoptosome assay. The apoptosome havingmutant APAF1 is expected to display increased caspase activation ascompared to wild-type APAF1. Upon incubation of apoptosome having mutantAPAF1, compounds that reduce caspase activation are considered to bepotential depression therapeutics. In another configuration, theapoptosome can be reconstituted in the presence of a compound known toenhance caspase activation by increasing the activity of the apoptosome.Compounds known to activate apoptosis via the apoptosome are known anddisclosed in Nguyen et al. PNAS 100:7533 7538 (2003) and Jiang et al.Science 299:223 226 (2003). One such compound isα-(trichloromethyl)-4-pyridineethanol. The ability of test compounds toreverse the enhancement of activation is indicative of a drug candidatefor treating depression.

Again, any method can be used to determine the affect of test compoundsfor their ability to reconstitute a functional apoptosome. In onemethod, activity can be monitored by procaspase-9 conversion tocaspase-9 can assessed by SDS-PAGE analysis and western blot analysisusing anti-caspase-9. In another method, a caspase activation assay canbe used to determine if procaspase-9 is converted to caspase-9 byassaying for caspase-3 activity.

The apoptosome reconstitution assay that can be used in these studies issimilar that of Zou et al. J. Biol. Chem., (1999), 274(17):11549 11556.The assay involves incubating purified cytochrome C, recombinant APAF1or APAF1 mutants, recombinant procaspase-9, and dATP, in a buffer having20 mM HEPES, 10 mM KCl, 2.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, pH7.5 at 30° C. After a set amount of time, the amount of conversion ofprocaspase-9 to caspase can be estimated using SDS-PAGE and western blotanalysis. Additionally, caspase activation (indicative of procaspase-9to caspase-9 by the apoptosome) can be measured using the caspasesubstrate Ac-LEHD-pNA (Upstate Biotechnology, NY) in a buffer having 50mM HEPES, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, 10 mM DTT,100 mL total volume, 15 mL apoptosome reconstitution reaction. Thereaction progress can be monitored at .DELTA.A405 nm.

In summary, compounds capable enhancing activation of the mutant APAF1apoptosomes in the apoptosome activation assay as compared to wild-typeAPAF1 are considered drug candidates for therapeutically treatingdepression.

Example 4 Screen of APAF1 Oligimerization Disrupters

Drug candidate for treating depression can be identified by screeningfor molecules that disrupt the oligimerization of APAF1. According tothis example, a truncated version of APAF1, which is constitutivelyactive, can be used to identify potential depression therapeutics. Thetruncated version of APAF1 can be any APAF1 as long as it isconstitutively active. Truncated APAF1 is disclosed in Srinivasula etal. Mol. Cell. 1 949 957 (1998), Hu et al. EMBO J. 18, 3586 3595 (1999),or according to SEQ ID NO:3 as disclosed herein. The truncated APAF1 canalso have any of the mutations disclosed herein.

Assay solution having truncated APAF1 and procaspase-9 is incubated inthe presence and absence of test compound. Test compounds which reducethe level of procaspase-9 (or procaspase-3) activation as compared to anidentical assay without the test compound is considered a potentialdepression therapeutic. Procaspase-9 and/or procaspase-3 activation canbe determined according to the examples described herein. The skilledartisan is capable of determining procaspase-9 and/or procaspase-3activation.

Example 5 Secondary Screen of Apoptosome Disrupters in Animal DepressionModels

Test compounds identified in the screens of the invention as potentialdepression therapeutics are desirably further tested in an animaldepression model. The potential depression therapeutics can be tested intransgenic animals being homozygous or heterozygous for a mutant APAF1.For example a transgenic animal being homozygous or heterozygous for amutant APAF1 well display a certain phenotype, animal treated withpotential depression therapeutics that are capable of modifyingdepression are expected to modify that phenotype. Thus, a group ofuntreated animals can be compared to a group of animal treated with thepotential depression therapeutic. The treated group is expected todisplay an improved phenotype. Any phenotypic measurement known to theskilled artisan can be used to assess the treatment.

Alternatively, the test compounds identified in the screens of theinvention as potential depression therapeutics are desirably tested inan art-accepted animal depression model such as those in Willner, Adv.Biochem. Psychopharmacol. 49:19 41 (1995); Porsolt, Rev. Neurosci. 11:5358 (2000); and Nestler et al. Neuron 34:13 25 (2002). These tests caninclude the forced swim test, the tail suspension test, learnedhelplessness test, chronic mild test stress, social stress test, earlylife stress test, olfactory bulbectomy test, fear conditioning test,anxiety based tests, reward based-tests, and cognition tests. Apotential depression therapeutic identified in the screens of theinvention has anti-depression activity if it increases the struggle timein the forced swim test, increases struggle time in the tail suspensiontest, decrease escape time and latency in the learned helplessness test,increased sexual behavior or sucrose preference in the chronic mildstress test, decrease behavioral abnormalities in the social stresstest, reverse behavioral problems in the early life stress test,reverses behavioral abnormalities in the olfactory bulbectomy test,decreases fear-like response when exposed to previously neutral cuesthat have been associated with adversive stimuli, increase the degree towhich an animal explores a particular environment in an anxiety basedtest, and so on.

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated herein byreference.

1. A method for detecting susceptibility to depression in an individualcomprising: drawing a sample of DNA from any tissue in the individual;and using a diagnostic technique to detect any alteration of thewild-type APAF1 allele.
 2. The method according to claim 1, wherein thediagnostic technique is selected from the group consisting offluorescent in situ hybridization (FISH), direct DNA sequencing,pulsed-field gel electrophoresis (PFGE) analysis, Southern blotanalysis, single stranded conformation analysis (SSCA), denaturinggradient gel electrophoresis (DGGE), RNase protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, allelespecific polymerase chain reaction (PCR), DNA microchip technology, andPCR-SSCA.
 3. The method according to claim 1, wherein the diagnostictechnique includes molecular cloning of the APAF1 allele(s) andsequencing the allele(s).
 4. The method according to claim 1, whereinthe diagnostic technique includes amplifying the gene sequences directlyfrom a genomic DNA preparation.
 5. The method according to claim 1,wherein the diagnostic technique includes directly comparing genomicAPAF1 sequences from the individual with those from a controlpopulation.
 6. The method according to claim 1, wherein the diagnostictechnique includes sequencing mutant alleles to identify the specificmutation for each allele.
 7. The method according to claim 1, whereinthe diagnostic technique includes: providing a probe/primer comprised ofan oligonucleotide which hybridizes to a sense or antisense sequence ofthe APAF1 gene or naturally occurring mutants thereof, or 5′ or 3′flanking sequences naturally associated with the APAF1 gene; contactingthe probe/primer to an appropriate nucleic acid containing sample; anddetecting, by hybridization of the probe/primer to the nucleic acid, thepresence or absence of a polymorphism.
 8. The method of claim 7, whereindetecting the polymorphism comprises utilizing the probe/primer todetermine the nucleotide sequence of an APAF1 gene and, optionally, ofthe flanking nucleic acid sequences.
 9. A method for screening testcompounds to identify antagonists or agonists of an APAF1 proteincomprising: contacting an agent with an APAF1 polypeptide, or homolog,derivative, or fragment thereof; and assaying (i) for the presence of acomplex between the agent and the APAF1 polypeptide, homolog,derivative, or fragment thereof, or (ii) for the presence of a complexbetween a ligand and the APAF1 polypeptide, homolog, derivative, orfragment thereof.
 10. The method according to claim 9, wherein the APAF1polypeptide or fragment is labeled.
 11. The method according to claim 9,wherein the APAF1 polypeptide, or homolog, derivative or fragmentthereof is a wild-type APAF1.
 12. A method of screening for a substancethat modulates the activity of a polypeptide comprising: contacting oneor more test substances with the polypeptide in a suitable reactionmedium; testing the activity of the treated polypeptide; and comparingthe activity of the treated polypeptide with the activity of a similarpolypeptide in comparable reaction medium untreated with the testsubstance or substances.
 13. The method according to claim 12, whereinthe polypeptide is an APAF1 polypeptide.
 14. A method of screening forinhibitors that disrupt the interaction between APAF1 or wild-type APAF1and its interacting partners comprising: contacting a test compound witha solution comprising the APAF1 or wild-type APAF1, cytochrome c, eitherATP or dAPT, and either procaspase-9 or procaspase-3; incubating thesolution; and analyzing the solution for the formation of eithercaspase-9 or caspase-3.
 15. A method for modulating the transcription ofcertain genes in a cell comprising: assaying to identify modulators ofAPAF1 or an altered form thereof; forming the modulators intotherapeutically effective or prophylactically effective drugs.
 16. Anisolated altered APAF1 polypeptide comprising: the polypeptide of SEQ IDNO:2; wherein the polypeptide as at least one of the following: (a) theCys at position 450 substituted with Trp; (b) the Gln at position 465substituted with Arg; (c) the Glu at position 777 substituted with Lys;(d) the Asn at position 782 substituted with Thr; (e) the Thr atposition 953 substituted with Ala; (f) the Leu at position 415substituted with Pro; (g) the Ser at position 357 substituted with Leu;(h) the Asp at position 479 substituted with Glu; or (i) the Glu atposition 625 substituted with Ala.
 17. A protein molecule comprising theamino acids set forth in SEQ ID NO:3.
 18. A method for detecting analteration in the APAF1 gene that is associated with depression in ahuman comprising: identifying a phenotype of people having a high riskof depression; drawing samples of DNA from the people within thephenotype; and screening the DNA to narrow the region containing thealteration.
 19. The method according to claim 18, wherein the phenotypecomprises people having major depression or bipolar disorder.
 20. Themethod according to claim 18, wherein the phenotype consists of maleshaving a high risk of depression.
 21. The method according to claim 18,wherein the phenotype comprises multiple ethnic populations.
 22. Themethod according to claim 18, wherein the screening comprises examiningvariants in the gene that are common among individuals within thephenotype.
 23. A method for determining whether a human subject has oris at risk for developing depression comprising: drawing a DNA samplefrom the human; and screening the APAF1 gene for the presence ofwild-type APAF1.
 24. The method according to claim 23, wherein thescreening step comprises ascertaining the existence of at least one of:a deletion of one or more nucleotides; an addition of one or morenucleotides; a substitution of one or more nucleotides; a grosschromosomal rearrangement; an alteration in the level of a messenger RNAtranscript; the presence of a non-wild type splicing pattern of amessenger RNA transcript; a non-wild type level of an APAF1 protein;and/or an aberrant level of an APAF1 protein.
 25. The method accordingto claim 23, wherein the examination of the APAF1 gene comprisesexamining whether any of the following substitutions are present: (a)the Cys at position 450 substituted with Trp; (b) the Gln at position465 substituted with Arg; (c) the Glu at position 777 substituted withLys; (d) the Asn at position 782 substituted with Thr; (e) the Thr atposition 953 substituted with Ala; (f) the Leu at position 415substituted with Pro; (g) the Ser at position 357 substituted with Leu;(h) the Asp at position 479 substituted with Glu; or (i) the Glu atposition 625 substituted with Ala.
 26. A method of screening for drugcandidates useful in treating depression comprising: drawing a DNAsample from the human; and screening the APAF1 gene for the presence ofwild-type APAF1.